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Developing a bioluminescence tracking system targeting on tumor cells and T cells for cancer immunotherapy

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Developing a bioluminescence tracking system targeting on tumor cells and T cells for cancer immunotherapy

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Content Developing a bioluminescence tracking system targeting on tumor cells and T cells for cancer
immunotherapy
By
Pin Yu Hou
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements of the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
Ma y 2023
Copyright 2023 Pin Yu Hou
Dedication
To my dear family, friends for their support and love.
ii
Acknowledgements
I would like to first appreciate my mentor Dr. Alan Epstein for accepting me to join his
laboratory, guiding me and providing me not only technical knowledge but also mental support
to complete the research and my thesis.
Besides my mentor, I would also like to thank Dr. Harvey Kaslow for organizing lab meeting and
providing me lots of resource regarding thesis writing, Dr. Peisheng Hu for helping me with the
difficulties I’ve meet when doing molecular cloning, and Dr. Leslie Khawli for coordinating all
the lab issues.
I also want to express my thank to Dr. Weiming Yuan for being my thesis defense committee
chair, and Dr. Keigo Machida, Dr. Harvey Kaslow for being my committee members.
Lastly, I want to specially thank my colleagues in Dr. Alan Epstein’s lab, including Chumeng
Chen, Vyshnavi Pachipulusu, Yekta Rahimi, Cailyn Lee, Nicole Elhosni, Olivia Hart. Without
their accompany and support, I won’t be able to complete my research.
iii
Table of contents
Dedication.......................................................................................................................................ii
Acknowledgements........................................................................................................................iii
Table of Figures...............................................................................................................................v
Abstract..........................................................................................................................................vi
Main section
Chapter 1: Introduction.................................................................................................................1
Chapter 2: Materials and Methods................................................................................................6
Chapter 3: Results.......................................................................................................................14
Chapter 4: Discussion and Future Directions.............................................................................25
Reference......................................................................................................................................27
iv
Table of Figures
Figure 1: The basic structure of CAR constructs.............................................................................2
Figure 2: The graph of bioluminescence generation........................................................................3
Figure 3: Schematic of the experiment design of this study............................................................5
Figure 4: The plate design and the time line of the in vitro assays................................................11
Figure 5: The time line of the animal experiments........................................................................13
Figure 6: Structure of Rluc and Fluc vectors.................................................................................14
Figure 7: Flow cytometry results of Rluc and Fluc transduction..................................................16
Figure 8: Flow cytometry results of puromycin selection.............................................................18
Figure 9: Rluc signal degradation curve of SKOV3, U251, and NT2.5LM..................................19
Figure 10: Bioluminescence results of in vitro assays...................................................................21
Figure 11: Imaging results of animal experiments.........................................................................23
v
Abstract
Bioluminescence has been a widely-used technique in molecular and cell biology. In the
research field of cancer therapy, it also serves as a practical tool to observe tumor growth and
evaluate cytotoxicity of T cells. In this research, our main aim is to develop a bioluminescence
tracking system that will enable us to monitor tumor cells and T cells simultaneously. Two
different categories of luciferase were used in this study: firefly luciferase (Fluc) and Renilla
luciferase (Rluc). Fluc vector was transduced to Jurkat cells and T cells while Rluc vector was
transduced to three different tumor cell lines. Both in vitro and in vivo assay were done to assess
the feasibility of this system. In in vitro assay, Rluc-transduced tumor cells were co-cultured with
Fluc-transduced Jurkat cells in a 96-well ELISA plate, followed by bioluminescence evaluation
via an ELISA reader (BioTek Synergy Microplate Reader). Animal experiments were done by
injecting Rluc-transduced tumor cells and Fluc-transduced Jurkat cells into mice, and then
imaging in vivo bioluminescence activity using a Xenogen IVIS 200 system. From these studies,
the activity of luciferase in tumor cells and Jurkat cells has been assessed, and a comprehensive
bioluminescence imaging process has been developed. It is hoped that the expression of
luciferase in CAR T cells and the application of this method to track these cells in vivo will
enable investigators to optimize the use of these cells for the treatment of cancers and related
diseases.
vi
Chapter 1: Introduction
The development of cancer immunotherapy
Cancer therapy was brought into a new era over the past decade by the development of cancer
immunotherapy, especially by the use of immune checkpoint blockade and chimeric antigen
receptor T cells (CAR T cells)
[1][2]
. The concept of CAR was first purposed by Zelig Eshhar and
colleagues in 1993, but it took over fifteen years of research to bring this technology in to the
clinic as a novel and successful method of cancer treatment. Briefly, T cells from patients were
transduced with antigen receptor genes encoding a single chain antibody, a transmembrane
domain, a co-stimulation domain, and a signal domain (Figure 1). With the engineered receptor
expressed, T cells not only acquire stronger affinity to recognize tumor cells, but also improve
the persistence and activity of T cells
[3]
. CAR T therapy was first clinically applied on
hematologic neoplasms including acute lymphoblastic leukemia (ALL)
[4]
and lymphoma
[5]
. The
application of CAR T cells against solid tumors, however, still remains a challenge due to several
causes including correct trafficking of CAR T cells to migrate to and enter the tumor site where
they encounter a highly suppressive tumor microenvironment
[6]
. Therefore, a systematic method
to assess the efficacy of CAR T cells is critical for the research to progress to overcome these
observed obstacles.
1
2
Figure. 1
The basic structure of CAR constructs. There are three main domains of CAR:
extracellular binding domain consisting of a single chain antibody and linker which is
responsible for binding with tumor cells, a transmembrane domain and an intracellular
co-stimulatory and signal domains, which are responsible for the stimulation of T cell
activation and cytotoxicity.
The development of bioluminescence
The term bioluminescence was first used in 1916
[7][8]
, which depicts the cold light generated
from living organisms. Within the generation of bioluminescence, luciferase serves as an
essential element to facilitate the process. Briefly, luciferase is defined as a category of enzymes
which oxidizes a substrate to produce a bioluminescence emission
[9]
(Figure 2). Various types of
luciferase have been identified in different organisms, including firefly luciferase, Renilla
luciferase, Gaussia luciferase as examples. Each luciferase targets a different substrate to
generate bioluminescence
[10][11]
. Because of its high sensitivity and low background signal,
bioluminescence has been a versatile tool in molecular and cell biology fields
[12][13]
.
3
Figure. 2
The graph of bioluminescence generation. With the presence of luciferase, substrates
are catalyzed to be oxidized. This chemical reaction is accompanied by the generation of
luminescence or light.
The application of bioluminescence on CAR T therapy
The technique of bioluminescence also serves as a good tool in CAR T therapy research,
providing a longitudinal, non-invasive technique to observe tumor development
[14][15]
, T cells
accumulation
[16]
, and the cytotoxicity of engineered CAR T cells
[10][17]
. Even though using a
single type of luciferase to track either tumors or T cells is a widely-used technique to study
cancer immunotherapy, the application of using multiple luciferases at the same time still
remains limited. Therefore, as an extension of previous studies, developing a new tracking
system by using two different types of luciferase to simultaneously monitor tumor growth and T
cell distribution was our main purpose of this research. To complete these studies, firefly
luciferase (Fluc) and Renilla luciferase (Rluc) were the two main luciferases used in this
research. Firefly luciferase was first isolated from a firefly named Photinus pyralis. With the
presence of the substrate D-luciferin, Fluc can catalyze the oxidation of D-luciferin and generate
green light. Similarly, Renilla luciferase was the one found in sea pansies uses coelenterazine as
its substrate, which can be oxidized to produce a blue light emission (Table 1). Briefly, in this
research, Fluc and Rluc vectors were designed to be transduced into T cells and tumor cells,
respectively. By injecting both Fluc-transduced T cells and Rluc-transduced tumor cells into
mice, two different bioluminescence signals can be detected through in vivo imaging. In addition
to in vivo bioluminescence imaging, in vitro assays were also designed to establish an optimal
imaging process in this study (Figure 3).
4
Table 1. Comparison between Firefly luciferase and Renilla luciferase
5
Figure. 3
Schematic of the experiment design of this study. Fluc and Rluc vectors were
transduced into T cells and tumor cells, respectively, followed by in vitro and in vivo
assays to establish the efficacy of this imaging system.
Chapter 2: Materials and Method
Cells
HEK 293T cells, SKOV3, U251, and Jurkat cells were all obtained from ATCC. HEK 293T cells
were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% of non-essential
amino acid, 1% of GlutaMAX, 1% of penicillin/streptomycin. SKOV3, U251, and Jurkat cells
were cultured in RPMI-1640 with 10% FBS and antibiotics. NT2.5LM cells were kindly
provided by Dr. Evanthia Roussos Torres’s lab at the University of Southern California and
cultured in RPMI-1640, supplemented with 20% FBS, 1% 1M HEPES, 1% penicillin/
streptomycin, 2% 200mM l-glutamine, 1% nonessential amino acid, and 1% 100mM sodium
pyruvate. T cells were obtained from patients and isolated by EasySep Human T cell Isolation
Kit following the manufacturer’s protocol and cultured in T cells media (43% Clicks’s medium,
43% RPMI-1640, 10% dFCS, 2% GlutaMAX, 1% nonessential amino acid, and 1% penicillin/
streptomycin) supplemented with 50ng/ml of rhIL-15 and rhIL-7. Rluc-transduced SKOV3,
U251, NT2.5LM cells, and Fluc-transduced Jurkat cells were developed during this research.
SKOV3-Rluc, U251-Rluc, and NT2.5LM-Rluc were cultured in the above cell media
supplemented with 10ug/ml of puromycin. Jurkat-Fluc was cultured in RPMI-1640
supplemented with 10% FBS and 5ug/ml of puromycin.
Mice
Animal experiments were approved by the USC Animal Care and Use Committee. NOD Scid-
IL2R
gammanull
(NSG) mice involved in this research were either purchased from Jackson
Laboratories or bred in the USC animal facility.
6
Reagents
Reagents used to complete this research included: RPMI-1640 (Genesee Scientific, REF#
25-506), DMEM (Genesee Scientific, REF# 25-500), phosphate-buffered saline (Corning, REF#
21-040-CV), fetal bovine serum (Omega Scientific, Catalog# FB-01), penicillin-streptomycin
(Genesee Scientific, REF# 25-512), l-glutamine (Genesee Scientific, REF# 25-509), GlutaMAX
(Thermo Fisher Scientific, Catalog# 35050-061), nonessential amino acids (Genesee Scientific,
REF# 25-536), HEPES (Sigma Aldrich, Product# H-7006), NucleoBond xtra midi prep kit
(Takara Bio, Catalog# 740410.50), Polyjet DNA in vitro transduction reagent (SignaGen
Laboratories, Catalog# SL100688), psPAX2 (Addgene, Catalog# 12260), pMD2.G (Addgene,
Catalog# 12259), LentiBlast Premium (OZ Biosciences, Catalog# LBPX500), puromycin
(InvivoGen, Catalog# ant-pr-1), coelenterazine (GoldBio, Catalog# CZ2.5), UbC-Luciferase-
RFP-TK Lentivector (System Bioscience, Catalog# BLIV202PA-1), pLenti-PGK-Venus-Fluc
(puro) (Addgene, Catalog# 140328), and ImmunoCult
TM
Human CD3/CD28/CD2 T-cells
Activator (Stemcell
TM
Technologies, Catalog# 10970).
Vector preparation
Fluc and Rluc vector were purchased from Addgene and System Bioscience respectively as
described above. Both vectors bear the puromycin resistance gene to enable cell selection.
Vectors were directly transformed to competent E.coli cells and streaked on LB agar plate.
Colonies were then picked for preparing midi prep by using NucleoBond xtra midi prep kit as
described by the manufacturer. Plasmid products were stored at -20℃ until further use.
7
Virus production
Transfection of HEK 293T cells was conducted to produce lentivirus by following Polyjet
TM
in
vitro DNA Transfection Reagent protocol. Transfection was conducted when HEK 293T cells
reached 90% confluency in T175 flask. Vectors were transduced to HEK 293T cells along with
psPAX2, pMD2.G (mass ratio 2:1:1) and Polyjet
TM
reagent (104ul/per T175 flask). Medium was
replaced 24 hours post-transfection. Supernatant containing lentivirus particles was collected 48
hours and 72 hours post-transfection, at which time cell debris was removed by centrifugation
and filtration through 0.45 µm polyethersulfone (PES) membrane. The filtrate was concentrated
by ultracentrifugation at 20,000*g for 2 hours, after which pelleted virus was resuspended in
PBS supplemented with 10% BSA. Collected lentivirus was stored at -80℃ until further use.
Luciferase transduction and analysis
Tumor cell line transduction and analysis
SKOV3, U251, and NT2.5LM cells were transduced with Rluc lentivirus. For this procedure,
0.5*10
6
cells per well were seeded in a 24 well plate, followed by adding 10ul 1M HEPES, 5ul
Lentiblast, three different volume of virus (20ul, 10ul, 2ul), and cell media to make the final
volume per well 500ul. Similarly, Jurkat cells were transduced with Fluc lentivirus as per the
method same with Rluc transduction. No virus was added to control groups. The plate was
centrifuged at 800*g in 32℃ for 2 hours, then incubated in 37℃. Media was replaced 24 hours
post-transduction. After 72 hours of incubation, transduced cells were washed twice, resuspended
in 500ul of PBS, and subjected to flow cytometry. Transduction efficiency was evaluated by
using an Attune flow cytometry from Thermo Fisher Scientific.
8
T cells transduction
Isolated T cells were cultured in T cell media and activated by adding 20ul of ImmunoCult
TM

Human CD3/CD28/CD2 T-cells Activator to 0.1*10
6
T cells in volume of 100ul three days
before transduction. On day 0, a 24 well plate was coated with 7ul retronectin
(concentration=1ug/ul) in 500ul PBS per well, then incubated in 4℃ overnight. On day 1,
retronectin-PBS solution was removed and the wells were blocked by using 2% BSA for 30
minutes. To each well, 20ul Fluc lentivirus, 10ul 1M HEPES, 5ul Lentiblast, and T cell media to
make total volume 250ul were added, followed by centrifugation at 2000*g at 4℃ for one hour.
No virus was added to the control group. Then, 0.5*10
6
of T cells in 250ul were added to the
virus mixture, and centrifuged again at 400*g at 32℃ for 45 minutes. The plate was incubated at
37℃ and the medium was replaced 24 hours post-transduction. Fluc expression was evaluated
on Attune flow cytometry (Thermo Fisher Scientific) 3 days after transduction.
Luciferase-transduced cells selection
Cell selection was conducted in the presence of puromycin. Working concentration for each cell
line was first tested by treating Non-transduced cells with 50ug/ml, 20ug/ml, 10ug/ml, or 5ug/ml
of puromycin-containing medium. Fresh cell media was added to control group. The lowest
concentration which killed all the non-transduced cells around the third day was determined to be
working concentration. Transduced cells were first expanded in T175 flask until confluency
reached 90%. Selection was then conducted by replacing fresh media with puromycin-containing
cell medium. For SKOV3-Rluc, U251-Rluc, and NT2.5LM-Rluc, cell media supplemented with
puromycin at the concentration of 10ug/ml was used. For Jurkat-Fluc, concentration of 5ug/ml
9
was used instead. At 72 hours post-selection, viable cells were expanded and frozen in liquid
nitrogen tank until further use.
In vitro bioluminescence assay of Rluc and Fluc transduced cells
Coelenterazine working concentration testing
0.1*10
6
Rluc-transduced cells in 150ul were seeded per well in ELISA 96 well plate. Non-
transduced cells were used as the mock group. At 24 hours after seeding, medium was replaced
with 4-fold series dilutions of coelenterazine-containing cell media. Rluc signals were then
detected every 10 minutes using the 460/40 channel on a BioTek Synergy Microplate Reader.
Bioluminescence assay
In ELISA 96 well plate, five testing groups were designed: Group 1 was mock cells, Group 2 was
luciferase transduced cells, Group 3 was Rluc transduced tumor cells cultured with mock Jurkat
cells, Group 4 was mock tumor cells cultured with Fluc-transduced Jurkat cells, and Group 5 was
Rluc-transduced tumor cells cultured with Fluc-transduced Jurkat cells (Figure 4A). For Group 1
and Group 2, 0.1*10
6
cells in 150ul were seeded in each well. For the other three groups, 0.1*10
6

cells for each cell type were seeded, making 0.2*10
6
cells in 150ul in total per well. Twenty-four
hours post-seeding, in vitro assay was conducted by testing Fluc expression first, followed by
examination of Rluc expression
[18]
. For testing Fluc expression, 100ul luciferin at a concentration
of 150ug/ml was added to wells containing mock Jurkat cells and Fluc-transduced Jurkat cells,
followed by incubation in dark room at room temperature for 10 minutes. Bioluminescence
activity was tested using a 528/20 channel on the BioTek Synergy Microplate Reader. Twenty
minutes after Fluc measurement, Fluc expression was examined again with the channel of
10
460/40, which was used to evaluate Rluc expression in order to confirm no Fluc signal was
detected. Rluc expression was performed by adding coelenterazine to wells containing mock and
Rluc-transduced SKOV3, U251, and NT2.5LM, immediately followed by Rluc measurement on
the BioTek Synergy Microplate Reader via 460/40 channel (Figure 4B). According to the results,
SKOV3 and NT2.5LM had a coelenterazine final working concentration of 37.5nM, and U251
has a final working concentration of 9.375nM. The bioluminescence activity of Fluc and Rluc
were detected by 528/20 and 460/40 channels on the ELISA reader, respectively.
11
(B)
(A)
Bioluminescence expression examination in NOD Scid-IL2R
gammanull
(NSG) mice
1.5*10
6
SKOV3-Rluc, U251-Rluc, and NT2.5LM-Rluc cells in 100ul PBS were injected
intraperitoneally to each NSG mouse using a 1ml syringe and 25 gauge needle. The same amount
of non-transduced cells were injected as mock groups (injection day defined as day 0).
Luciferase expression was assessed on day 5. For these studies, mice were anesthetized with
vaporized isoflurane and treated with 100ul of 1ug/ul coelenterazine via intravenous injection
immediately before subjected to bioluminescence imaging
[19]
. On day 6, 4*10
6
of Jurkat-Fluc
[20]

were injected via the tail vein to mice and then subjected to in vivo imaging again on day 7. Non-
transduced Jurkat cells were injected as the mock group. Similarly, after anesthesia, mice were
administered with D-luciferin via intraperitoneal injection and imaged for Fluc expression after
10 minutes. Likewise, 100ul of 1ug/ul coelenterazine was injected intravenously, followed by
imaging for Rluc signal. All in vivo bioluminescence imaging in this research were conducted
using an Xenogen IVIS 200 at the USC Molecular Imaging Center (Figure 5).
12
Figure 4.
The plate design and the time line of the in vitro assays.
(A) The plate design of in vitro assay. Group 1 and Group 2 were the mock and
luciferase-transduced form of each cell line. Group 3 was Rluc-transduced tumor cells
cultured with mock Jurkat cells. Group 4 was mock tumor cells cultured with Fluc-
transduced Jurkat cells. Group 5 was Rluc-transduced tumor cells and Fluc-
transduced Jurkat cells cultured together.
(B) Time line of the in vitro assay. Fluc signal was tested with 528/20 channel on ELISA
reader 10 minutes after adding D-luciferin, and tested again after 20 minutes with
460/40 channel. Coelenterazine was then added, immediately followed by Rluc signal
evaluation through 460/40 channel.

13
Figure 5.
The time line of the animal experiments. Rluc-transduced human tumor cell lines
SKOV3-Rluc, U251-Rluc, and NT2.5LM-Rluc were firstly injected to mice. Five days
after injection, first imaging was performed to assess Rluc activity. On day 6, Jurkat-
Fluc was then injected to mice and Fluc expression was then imaged next day after
injection.
Chapter 3: Results
Selection of Rluc and Fluc vectors
Commercial Firefly luciferase (Fluc) and Renilla luciferase (Rluc) vectors were used in this
research. In order to facilitate stable luciferase-transduced cell lines development for further use,
purchased vectors both contained puromycin resistance sequences which enable the selection of
positive cells. Additionally, both Rluc and Fluc vectors bear sequences of fluorescent proteins,
which can be detected by flow cytometry for transduction evaluation, including EGFP and Venus
(a type of GFP) on Rluc and Fluc vector, respectively. After transfection, purified plasmid
products were outsourced to Genewiz for sequencing (Figure 6).
14
Figure 6.
Structure of Rluc and Fluc vectors. Besides the respective luciferase sequence, both
Rluc and Fluc vectors contain GFP (EGFP and Venus) and puromycin resistance
sequences.
Luciferase transduction
Renilla luciferase (Rluc) transduction on human tumor cell lines SKOV3, U251 and
NT2.5LM
Rluc transduction efficiency on SKOV3, U251 and NT2.5LM cells was assessed by flow
cytometry three days post-transduction. The results demonstrate that the EGFP signal from these
three tumor cell lines were all detected, which was an indicator to guarantee Rluc vectors were
successfully transduced into tumor cells. With the maximum transduced lentivirus volume
(20ul), SKOV3 and U251 both reached over 85% expression, while NT2.5LM also had 64.3%
expression. Additionally, for both SKOV3 and U251, the expression levels showed increased
levels with larger transduced virus volumes. Since transduction of NT2.5LM was conducted after
the other two cell lines, the maximum volume of virus was directly applied to NT2.5LM
transduction (Figure 7A).
Firefly luciferase (Fluc) transduction on Jurkat cells and T cells
Jurkat cells and human T cells were used as target cells of Fluc transduction. In addition,
transduction efficiency was evaluated by detecting GFP signal by flow cytometry three days after
transduction. For Fluc transduction of Jurkat cells, with the maximum transduced virus volume
(20ul), Fluc expression rate was about 5%. Even though the average expression level was
relatively low, positive correlation still existed between the volume of transduced virus and the
Fluc transduction efficiency. For the Fluc transduction on T cells, only the group with maximum
virus volume was conducted, which produced an expression percentage of 40.5 (Figure 7B).
15
16
(A)
(B)
17
Figure 7.
Flow cytometry results of Rluc and Fluc transduction.
(A) Flow cytometry results of Rluc transduction on SKOV3, U251, and NT2.5LM human
cell lines.
(B) Flow cytometry results of Fluc transduction on Jurkat and human T cells.
Rluc vector was expressed on SKOV3, U251 and NT2.5LM, while Fluc expression on
Jurkat was relatively lower than the other groups. However, the positive correlation
between transduced virus amount with luciferase expression level was shown in every
group.
Establishment of Rluc and Fluc transduced cells
Puromycin was used for cell selection. The working concentration of puromycin was first
determined before selection. For non-transduced SKOV3, U251, and NT2.5LM, the cells seeded
in 24-well plates died three days after treatment with 10ug/ml of puromycin. For mock Jurkat
cells, all the cells were dead on the third day with 5ug/ml puromycin. These concentrations
became the working concentration and were then applied to future selections by culturing
luciferase-transduced cells in puromycin-containing cell media for three days. After selection,
transduced cells were subjected to flow cytometry which showed higher expression level (Figure
8). Selected cells were all expanded and passaged in order to ensure their stability before
freezing for long term storage in liquid nitrogen.
18
Figure 8.
Flow cytometry results of puromycin selection. Evaluation of luciferase expression
levels were performed again after puromycin selection. In all the cell lines, including
Rluc-transduced SKOV3, U251, NT2.5LM and Fluc-transduced Jurkat, the luciferase
expression levels were elevated compared to the results shown in figure 7.
Coelenterazine concentration testing
Coelenterazine is the substrate used to trigger Rluc expression. Since the amount of substrate
might affect the expression level of luciferase, appropriate working concentration of
coelenterazine for Rluc activation was tested in advance. In the results of SKOV3-Rluc, U251-
Rluc and NT2.5LM-Rluc all indicate that Rluc signal increases with the higher concentration of
coelenterazine treated. In order to attune the intensity of Rluc signal from each cell line as much
as possible, coelenterazine concentration of 37.5nM was chosen for SKOV3-Rluc, 9.375nM for
U251-Rluc and 75nM for NT2.5LM.
19
Figure 9.
Rluc degradation curve of SKOV3, U251 and NT2.5LM. Four different coelenterazine
concentrations were tested in this assay. In SKOV3, U251 and NT2.5LM, Rluc
expression showed increase with higher coelenterazine concentration. Final
coelenterazine decide to be used on the selection for each cell line is marked red.
37.5nM
In vitro bioluminescence assay
In this assay, five groups of cells were added to a 96-well plate. Fluc and Rluc were detected by
channel 528/20 and 460/40 on the ELISA reader, respectively. Both Jurkat-Fluc cultured alone in
the Group 2, and those co-cultured with tumor cells in Groups 4 and 5, the Fluc signals were all
detected. A comparison between Group 1 and Group 2, shown that the expression in this batch
was confirmed to be stable. Fluc signals were consistent in Group 4 and Group 5, indicating that
Fluc expression was not affected by the Rluc-transduced tumor cells. Twenty minutes later, Fluc
signals were examined again using channel 460/40 for Rluc expression, to confirm there is no
Fluc signal detected which might affect subsequent Rluc expression evaluations. Next, the
expression of Rluc was tested immediately by using channel 460/40 after adding coelenterazine.
All Rluc-transduced tumor cells in Group 2, 3, and 5 were found to have high expression level
compared to mock groups. Similarly, the results of Group 2 ensured the expression stability of
this batch of cells, and the comparable intensity of Rluc signal in group 3 and group 5 indicated
that the expression of Rluc was not affected by the existence of Fluc expression in these cells.
20
(A)

21
Figure 10.
Bioluminescence results of in vitro assays. (A) Fluc expression evaluation on Jurkat
cells via 528/20 channel. All Fluc-transduced Jurkat cells in each group expressed
Fluc signal compared to mock Jurkat. (B) Second Fluc expression examination by
using 460/40 channel. No Fluc signal was detected under 460/40 channel, which was
used to detect the Rluc signal. (C) Rluc was then tested via 460/40 channel.
Significant Rluc expression level in each group was shown in every group, regardless
of the existence of Fluc-transduced Jurkat.
(B)
(C)
In vivo bioluminescence of Rluc and Fluc in NSG mice
1.5*10
6
cells of U251-Rluc and NT2.5LM-Rluc were injected to mice intraperitoneally on day 0,
and Rluc activity was evaluated via Xenogen IVIS 200 at the USC Molecular Imaging Center on
day 5 (Figure 5). In comparison with the mock group, Rluc signals of U251-Rluc and NT2.5LM-
Rluc were both evident by in vivo imaging shown below (The left image in Figure 11A and
Figure 11B). After first imaging, 2*10
6
Jurkat-Fluc cells were then injected to mice via tail veins
on day 6. Similarly, mock Jurkat cells were injected to the mock groups. Second time in vivo
imaging was then performed on day 7 by testing Fluc signals first, followed by Rluc signals
assessment. For U251, both Jurkat-Fluc and U251-Rluc showed evident luciferase signals.
However, the location of Fluc and Rluc signals were partially overlapped, which possibly means
that the signals from Fluc and Rluc may have interfered with each other. Additionally, Rluc
signal of tumor cells on the second imaging did not match with the results from the first imaging
(Figure 11 A). For NT2.5LM, Fluc activity was only detected in one mouse, and was similar to
the results of U251. The Rluc signals of tumor cells in the first imaging were not detected in the
second imaging (Figure 11B). Because of these inconsistent results, a different imaging process
may need to be used to solve this problem in the future studies. Additionally, 2*10
6
Jurkat-Fluc
cells from the same batch were also injected alone via intravenous injection to mice and
subjected to imaging. In this study, Fluc signal was detected, especially on the tail, which was the
location of the injection site. With these results, the luciferase activity of this batch of Jurkat-Fluc
was verified (Figure 11C).
22

23
(B)
(A)
24
(C)
Figure 11.
Imaging results of animal experiments.
(A) The imaging results of U251. The left image showed the U251-Rluc activity tested
on day 5 after U251-Rluc injection, and the other two showed Fluc signal of Jurkat
cells and Rluc signal of U251 assessed on day 7. From these images, luciferase from
both U251 and Jurkat cells all showed strong activity.
(B) The imaging results of NT2.5LM. The left image showed the NT2.5LM-Rluc activity
imaged on day 5 after injection, and the right two showed Jurkat-Fluc and NT2.5LM-
Rluc signals, respectively, assessed on day 7. On day 5 imaging, Rluc signals of tumor
cells were strong. On day 7 imaging, however, Jurkat-Fluc was only detected in the
third mouse. Additionally, the NT2.5LM-Rluc signal of the third mice also not
detected.
(C) The imaging results of Jurkat cells. Jurkat-Fluc cells were injected on day 6 alone
in order to confirm the luciferase quality of this batch of Jurkat-Fluc, and imaging
was performed on the next day. Some signals located on the tail were detected
which at the injection site.
Chapter 4: Discussion and Future Directions
Both in vitro and in vivo bioluminescence imaging were examined in this research in order to
develop an effective protocol of testing Rluc and Fluc expression. However, there are still some
difficulties remain to be solved in the future. First, although Rluc activity in tumor cell lines were
strong in both in vitro and in vivo studies, Fluc activity in Jurkat cells, on the other hand, was
relatively weak, making it difficult to observe Fluc signal in the in vivo imaging. Despite the fact
that puromycin selection had already performed on Fluc-transduced Jurkat cells, the
improvement in luciferase expression rate was still limited. Hence, to generate a stable Jurkat
cell line with high Fluc bioluminescence activity, single cell sorting is now being performing on
the existing Fluc-transduced Jurkat cells in subsequent experiments. Once comparable luciferase
activity in tumor cells and Jurkat cells are attained, more precise imaging results can be
expected, which can further elevate the value of this tracking system. Additionally, since Fluc
transduction on human T cells produced a better transduction efficiency than Fluc transduction
on Jurkat cells, Fluc-transduced human T cells may be a better candidate for future studies.
However, human T cells have poor viability under puromycin treatment, an effective method to
select positive cells other than using puromycin may also be needed for these studies. Secondly,
unlike in vitro assays where specific wavelength targets for Fluc and Rluc can used by the ELISA
reader (BioTek Synergy Microplate Reader), the Xenogen IVIS 200 system used to performed in
vivo imaging can only detect bioluminescence signals with all wavelengths. Therefore, the issue
of whether Fluc and Rluc signal can affect each other remains to be determined in future
experiments. In the present study, solid tumor cell lines SKOV3, U251, and NT2.5LM were used
to transduce Rluc and additional studies will have to be done to determine if these methods will
work with human hematopoietic tumors. Lastly, For long term research purposes, the in vivo
25
study of Fluc-transduced human T cells and the application of this tracking system to CAR T
cells will be a major aim of future studies.
26
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28

Abstract (if available)

Abstract Bioluminescence has been a widely-used technique in molecular and cell biology. In the research field of cancer therapy, it also serves as a practical tool to observe tumor growth and evaluate cytotoxicity of T cells. In this research, our main aim is to develop a bioluminescence tracking system that will enable us to monitor tumor cells and T cells simultaneously. Two different categories of luciferase were used in this study: firefly luciferase (Fluc) and Renilla luciferase (Rluc). Fluc vector was transduced to Jurkat cells and T cells while Rluc vector was transduced to three different tumor cell lines. Both in vitro and in vivo assay were done to assess the feasibility of this system. In in vitro assay, Rluc-transduced tumor cells were co-cultured with Fluc-transduced Jurkat cells in a 96-well ELISA plate, followed by bioluminescence evaluation via an ELISA reader (BioTek Synergy Microplate Reader). Animal experiments were done by injecting Rluc-transduced tumor cells and Fluc-transduced Jurkat cells into mice, and then imaging in vivo bioluminescence activity using a Xenogen IVIS 200 system. From these studies, the activity of luciferase in tumor cells and Jurkat cells has been assessed, and a comprehensive bioluminescence imaging process has been developed. It is hoped that the expression of luciferase in CAR T cells and the application of this method to track these cells in vivo will enable investigators to optimize the use of these cells for the treatment of cancers and related diseases.

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Asset Metadata

Creator Hou, Pin Yu (author)

Core Title Developing a bioluminescence tracking system targeting on tumor cells and T cells for cancer immunotherapy

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2023-05

Publication Date 04/25/2023

Defense Date 03/21/2023

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bioluminescence,cancer immunotherapy,firefly luciferase,OAI-PMH Harvest,renilla luciferase,T cells,tumor tracking

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Yuan , Weiming (committee chair), Epstein, Alan (committee member), Kaslow, Harvey (committee member), Machida, Keigo (committee member)

Creator Email michaelj5520@gmail.com,pinyuhou5520@gmail.com

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