Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Assessment of theranostic agent for brain cancer
(USC Thesis Other)
Assessment of theranostic agent for brain cancer
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
Copyright 2020 Alesi Renee Escobedo
Assessment of Theranostic Agent for Brain Cancer
by
Alesi Renee Escobedo
A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2020
ii
Acknowledgements
I would like to thank Dr. Jean C Shih for giving me the opportunity to be part of her lab,
and the guidance she has given me throughout my time. I am thankful for her support and
teaching me how to improve my skills as a research, presenter, and role model for women in the
field of science.
I am thankful to senior research associate Dr. Ronald Irwin, for the teaching me the
importance of being open to suggestions, as well as being a supporter of my visionary mind in
research. I am grateful for the advice he has given me throughout my time in the lab, both
professional and personal advice, that has helped shaped the person I am today.
I would also like to thank Bin Qian for giving me important advice during the
experiments. Along with his assistance in both in vitro and in vivo experiments. I would also like
to thank the rest of my lab members, for creating a warm welcoming environment. Not to
mention being able to see our relationship evolve from colleagues to friends.
My greatest gratitude goes to my committee as well, for the support and feedback I have
received throughout this process.
Most importantly I want to thank my parents and sister for being there for me throughout
my journey. Thank you, guys, for celebrating with me when experiments were successful, thank
you for letting me vent to you when experiments didn’t go as planned or when I was going
through some tough times. Mom and Dad, I am grateful and forever indebted to you both for all
the sacrifices that you have made throughout this journey in order to help me succeed and
accomplish the goals I set for myself. Aryelle, thank you for being my reality check when I need
it, helping me not be so self-critical, and reminding me to also take time to enjoy the life that I
am living.
Por ultimo quiero dedicarle esta tesis a mi abuela. Abuela, que siempre me mostró cómo
ser una mujer fuerte y independiente. Tú siempre me has mostrado cómo seguir luchando, y
nunca perder la esperanza ni la fe cuando se enfrenta a tiempos dificlies. Tengo el honor de
llamarte mi abuela, me siento orgullosa de todo lo que tú has hecho para nuestra familia. Te amo
mucho!
iii
Table of Contents
Acknowledgements ................................................................................................................ii
List of Tables .........................................................................................................................iv
List of Figures ........................................................................................................................v
Abbreviations .........................................................................................................................vi
Abstract ..................................................................................................................................vii
Chapter 1: Introduction ..........................................................................................................1
Chapter 2: Materials and Methods .........................................................................................5
Chapter 3: Results ..................................................................................................................11
Chapter 4: Discussion and Conclusion .................................................................................23
References ..............................................................................................................................26
iv
List of Tables
Table 1: In vivo studies performed with NMI treatment on glioma ......................................13
v
List of Figures
Figure 1. Chemical Structure of TMZ, NMI, and Clorgyline ...............................................3
Figure 2. Synthesis of NMI ...................................................................................................6
Figure 3. NMI treatment reduces clonogenicity and growth of GL26 ..................................12
Figure 4. Biodistribution of NMI in C57BL wildtype mice. ................................................15
Figure 5. Specific uptake of NMI by tumor but not by normal tissue ..................................16
Figure 6. NMI Specifically targets cancer cells and shows a dose
response in mice and ex vivo ..................................................................................17
Figure 7. Body weight as a percentage of mice dosed with toxic levels of NMI .................19
Figure 8. High doses of NMI has minimal effect on exploration in mice ............................21
Figure 9. Gel of NMI bound to proteins ...............................................................................22
vi
Abbreviations
TMZ Temozolomide
NMI Near Infrared Dye MHI-148 Clorgyline
MAOA Monoamine Oxidase A
GBM Glioblastoma multiforme
FBS Fetal Bovine Serum
ROI Region of Interest
ROS Reactive Oxygen Species
SBECD Sulfobutylether-beta-cyclodextrin
HIF-1α Hypoxia Inducible Factor-1α
OATPs Organic Anion-Transporting Polypeptides
vii
Abstract
Brain cancer is universal, it can affect all people regardless of age, gender, or ethnicity.
Susceptibility for specific types of brain cancer varies. Glioblastoma multiforme (GBM) can be
deadly where the average survival time is 14-15 months from the time of diagnosis. Previous
work from this laboratory has shown that Near infrared dye MHI-148 conjugated Monoamine
oxidase Inhibitor clorgyline (NMI) is a therapy and diagnostic (theranostic), therefore it can both
treat and monitor the brain tumors. This is due to the targeting mechanism of NMI interacting
with a specific transporter in cancer cells. Using in vitro and in vivo brain tumor models have
shown of NMI to able to inhibit the development of glioma. NMI can be used as a theranostic for
glioma. This study shows that biodistribution, route of administration and the toxicity level of
NMI in the present and absence of brain tumor GL26 cells. When testing route of administration
both in non-tumor and tumor bearing mouse model, it was found that NMI is readily distributed
when administered intravenously, in comparison oral route of administration had minimal
distribution in the body indicating intravenous administration of NMI was to be used for optimal
biodistribution. Further biodistribution studies were conducted where the focus was on tumor
bearing mouse model. In a one-week study it was learned that NMI accumulates in the tumor and
not in the adjacent brain, peak accumulation at of NMI is 48h in tissue, however complete
clearance of NMI in blood is 48h, and elimination from tissue was more than 1 week. In addition
to NMI accumulation in the tumor and not in the adjacent brain, NMI follows the dose-response
relationship in that as NMI dose increases accumulation occurs, but at 50 mg/kg it reaches
maximum accumulation and is over saturated. Toxicology studies showed that at toxic levels of
NMI caused weight loss, but minimal effect on food consumption or exploration. In summary,
this study shows preferred route of administration, the safe and toxic dosages of NMI for the
viii
treatment and diagnosis of glioma. This new information provides important preliminary data for
future pharmacokinetic and other preclinical studies.
1
1. Introduction
Brain cancer is universal, it can affect all people regardless of age, gender, or ethnicity.
Susceptibility for specific types of brain cancer varies. Glioblastoma multiforme (GBM) can be
deadly where the average survival time is 14-15 months from time of diagnosis (Kushal et al.,
2016). Gliomas are slightly more likely to occur in men than in women, and more common in
Caucasians than in African Americans (John Hopkins, 2020). Patients experience headaches,
decline of brain function, memory loss, and personality changes; all of which significantly
diminishes their quality of life. GBM changes everything not only for the patients but for their
loved ones as well.
The current treatment options have been proven to be both limited and ineffective. For
example, surgery is not always an option due to limited accessibility to the tumor. As for
chemotherapy the current standard is Temozolomide (TMZ; Figure 1). TMZ is a cytotoxic
imidazotetrazine that leads to the formation of O
6
-methylguanine which mismatches with
thymine in DNA replication (D'Alessandro et al., 2016). Yet many tumors become resistant to
this treatment and grow at a more aggressive rate. The tumor microenvironment has a role in the
contribution towards the malignancy of cancer progression (Caja and Tan, 2019). The blood
brain barrier and the blood tumor barrier between tumor cells and micro vessels are major
obstacles to the delivery of agents to tumor tissues from systemic circulation (Wu et al., 2015b).
The majority of cancer therapeutics on the market today, are not suitable for glioma due to their
inability to cross the blood brain barrier and poor drug accumulation into the tumor tissue (Gao
et al., 2013).
There is an unmet and urgent need for the development of new therapeutics that can
effectively treat and monitor brain tumors. Near Infrared Dye MHI-148 Clorgyline (NMI) is a
2
therapy and diagnostic (theranostic), therefore you can both treat and monitor the brain tumors. It
is a compound that is composed of a monoamine oxidase A inhibitor and fluorescence dye. The
MAOA inhibitor component of NMI is Clorgyline (Figure 1) which is a selective and irreversible
inhibitor of MAOA. Near-infrared heptamethine carbocyanine dyes are a novel class of
heterocyclic polymethine cyanine analogues with cancer imaging ability due to their high
excitation coefficients and large Stokes shifts, with characteristic fluorescence emissions in the
range of 700-1000 nm (Yang et al., 2019). The fluorescence dye in the NIR range helps
minimize background interference and improve tissue penetration, depth, which allows for
sensitive and noninvasive imaging (Zhang et al., 2018). NMI works by targeting tumor cells due
to their overexpression of Monoamine Oxidase A (MAOA), as a result it accumulates in tumor
cells(Wu et al., 2015a).
The focus of this study is due to recent studies finding a link between tumorigenesis and
MAOA. MAOs are located at the mitochondrial outer membrane and catalyze the oxidative
deamination of monoamine neurotransmitters in the brain such as serotonin, norepinephrine,
dopamine phenylethylamine, and tyramine. MAO produces hydrogen peroxide (H2O2) as a
byproduct a source of reactive oxygen species linked to DNA damage and tumor progression.
(Shih, 2018). The ROS cause a hypoxic microenvironment which contributes to tumor
aggression and poor clinical outcome, this is due to hypoxic responses that cause cell
proliferation, survival, angiogenesis, and metastasis (Caja and Tan, 2019). Previous studies done
on NIR cyanine dyes showed that hypoxia in the tumor microenvironment induce the
preferential uptake of these dyes through the hypoxia inducible factor-1α/organic anion-
transporting polypeptides (HIF-1α/OATPs) which is expressed by various human cancers (Shih,
3
2018). OATPs facilitate the transport of substances into cells including organic acids, drugs, and
hormones (Wu et al., 2015b).
Fig.1 Chemical structure A) a chemical structure of TMZ, B) a chemical structure of NMI, C) a chemical structure
of Clorgyline.
The aim of this study using in vitro and in vivo brain tumor models is to investigate the
potential of NMI to inhibit the development of glioma. To test this hypothesis, we will develop a
formulation that can be applicable in clinical phases. Pharmaceutical formulation is the means by
which active principles of drugs are converted into preparations which are safe, effective and
convenient in use (Fishburn, 1965). The importance of formulation is to ensure the delivery
system of choice does not diminish the activity or safety of the drug. The discovery of optimal
formulation will be determined through studies related to pharmacokinetics focusing on blood,
tissue, and organ distribution over time. Biodistribution can be analyzed and tracked through the
use of imaging. Administered NMI will be studied by comparing oral gavage or intravenous
injection to determine which route of administration is optimal. To determine the efficacy and
potential adverse effects, dose range finding will be conducted using xenograft mouse models
approximately 3- to 6-months of age that received intracranial implantations of glioma cell lines.
The GL26 orthotopic tumor implantation to C57BL/6 mice has been used for more than 50 years
as an immunocompetent model suitable for assessing therapies and their possible interactions
4
with the host immune system (Ausman et al., 1970; Ferrer-Font et al., 2017; Sugiura, 1969).
Mice model are used as a way to predict starting dosages and identify potential side effects for
new drugs that will enter into early clinical trial, preclinical tolerability and safety pharmacology
studies in rodents and non-rodents are required (Aston et al., 2017). In the studies conducted, we
focused on weight loss, movement patterns, and other physical clinical signs related to toxicity.
At the end of each study, mice were imaged in vivo, subsequently tissues were collected and
imaged ex vivo. The tissue was then used in protein assays and other experiments to compare the
treatment groups.
5
2.Materials and Methods
Cells
Human glioma cell line U251 was obtained from American Type Culture Collection
(ATCC). Human glioma cell line U251R/S were cultured in 10% fetal bovine serum (FBS) in
Dulbecco’s Modified Eagle’s Media supplemented with 100 U/mL penicillin and 0.1 mg/mL
streptomycin in a humidified incubator at 37°C and 5% CO2. Glioma cell lines were originally
purchased from American Type Culture Collection (ATCC). Mouse glioma cell line GL26 was
cultured in 10% fetal bovine serum (FBS) in Dulbecco’s Modified Eagle’s Media supplemented
with 100 U/mL penicillin and 0.1 mg/mL streptomycin in a humidified incubator at 37°C and
5% CO2.
Reagents
NMI was synthesized previously (Kushal et al., 2016). The following drugs were used:
temozolomide (TMZ; Sigma Life Sciences). D-Luciferin Potassium Salt (Gold Bio). For in vitro
experiments TMZ was dissolved in DMSO and furthered diluted with culture medium to the
indicated concentrations. Luciferin was prepared fresh at 15 mg/mL in PBS. All compounds
were stored properly based on recommendations from the manufacturer. All the working
solutions were prepared fresh the day of experiment according to the protocol. NMI was
dissolved in a formulation containing 40% SBECD. For every 1 mg of NMI used, 400 mg of
SBECD was required, combine them and add 600 µL of ddH2O. Then vortexed (VWR Scientific
Products Mini Vortexer) then sonicated (Bronson Sonifier 450) with the following settings: duty
cycle 3, and output control 3. Sonication was done using an ice bath to control temperature
increase.
6
Fig 2: Synthesis of NMI (Wu et al., 2015a)
MAOA Catalytic Activity Assay
MAOA catalytic activity was determined by radio-assay described previously by (Kushal
et al., 2016). Briefly cells were incubated with 1 mM
14
C-5-hydroxytryptamine (5-HT) for 20
minutes, in the assay buffer at 37°C. The reaction was completed when 6 N HCl was added. The
reaction products were extracted using 1:1 ratio of benzene/ethyl acetate and then centrifuged.
The radioactivity was determined using the organic phase layer placed in a liquid scintillation
spectroscopy (Beckman Model LS 6000 IC Liquid Scintillation Counter).
MTT Assay
Glioma cells (7,500 cells per well) were seeded in 96-well plates. After 24 hours, TMZ,
or NMI, were added at different concentrations and the cells were incubated for 48 hours. The
assay was conducted according to the protocol. Absorbance was measured using a multimode
7
plate reader (Biotek Synergy HTX) at 570 nm. Percentage viability was calculated relative to
untreated control cells. All experiments were conducted in triplicates.
Colony-Forming Assay
Glioma cells were seeded in 6-well plates at 500 cells per well and allowed to adhere for
24 hours. After 24 hours, TMZ, or NMI, were added at different concentrations and the cells
were incubated for 48 hours. The drug treatment was then removed and fresh medium (without
drugs) added. Cell were incubated for an additional 8-10 days. At the end of the assay, colonies
were visualized by staining with crystal violet for 2 hours at room temperature, then washed with
water and dried overnight before being quantified. Groups were plated in triplicate. Images were
captured of the colony formation (The iBright FL1000 gel/cell imager), then analysis of colony
formation based on percentage of area covered in each well (ImageJ software).
Gel Electrophoresis
About 15 µg per well sample was loaded into each well of a 12% SDS-PAGE gels (Bio-
Rad Laboratories, Hercules, CA). Gels were electrophoresed with Tris/Glycine running buffer at
constant 100 voltage, for 2 hours. The gel was captured by iBright Imaging System
(ThermoFisher). All band intensities were quantified using iBright Analysis Software
(ThermoFisher).
Mice
Male C57BL wildtype mice were obtained from Charles River Inc. and housed in the
animal research facility at University of Southern California (USC). Mice were between 3 and 6
months of age, weighing 22-28 grams for these studies. All studies were conducted according to
the Institutional Animal Care and Use Committee of the University of Southern California
8
(IACUC) approvals (Protocol 20212). Mice were fed a normal diet; the animal facility
temperature was kept 22°C, and 14 hours of light followed by 10 hours of dark related to their
natural light cycle. Mice were monitored daily.
In vivo experiments
All animal protocols were approved by the Institutional Animal Care and Use Committee
of the University of Southern California (IACUC). Luciferase positive GL26 glioma cells (5,000
cells in 10 uL PBS injection) were implanted intracranially as previously described (Cho et al.,
2012). After implantation mice were randomly divided into groups, and treatment was initiated
based on Table 1 after implantation. Treatment is as follows: Control (SBECD), and NMI. NMI
was prepared and diluted under sterile conditions in SBECD and was administered intravenous
or oral gavage for a single dose. Mice are dosed intravenously (i.v.) using a 27G insulin syringe.
Mice are dosed orally using a 18G syringe gavage. Different experiments and groups were used
(Table 1). The therapy starting day was variable, based on the different experiments. Mice were
shaved prior to being imaged after treatment. Mice were imaged using IVIS Lumina Series III
(Perkin Elmer); equipped with fluorescence filter sets excitation/ emission, 780/845 nm (Wu et
al., 2015a). Images were analyzed using LIVING IMAGE software version 3.2 (Perkin Elmer).
After imaging, animals were euthanized by cervical dislocation according to the IACUC.
Bioluminescence Imaging
Mice were anesthetized with 2.5% isoflurane using an Inhalation Anesthesia System
(VetEquip, Inc., Pleasant Hill, CA) and were given a single intraperitoneal (i.p.) injection of
luciferin. Inject 10 µL/g of body weight (e.g. 20 g mouse, inject 200µL of 15 mg/mL stock
solution). After waiting for approximately 10 minutes to allow full systemic distribution of
9
luciferin, the mice were placed in the chamber of IVIS Lumina Series III (Perkin Elmer) with
continuous 2.5% isoflurane exposure. Images were captured with exposure of 5 seconds; other
settings were set according to the wizard option on the system.
Fluorescence Imaging
Mice were anesthetized with 2.5% isoflurane using an Inhalation Anesthesia System
(VetEquip, Inc., Pleasant Hill, CA). The mice were placed in the chamber of IVIS Lumina Series
III (Perkin Elmer) with continuous 2.5% isoflurane exposure. Imaging System settings for mice
are excitation 740 nm, emissions 790 nm, binning 8, field of view 24, f-stop 2, and exposure 1
second. Imaging System settings for organs are excitation 740 nm, emissions 790 nm, binning 1,
field of view 12.5, f-stop 2, and exposure 5 second.
Location Tracking
ezTrack’s location tracking assesses an animal’s location across the course of recorded
session (1 h post injection). Mounted camera is used so the field of view is fixed throughout the
session. Background contrast relative to mouse was set as the optimal working conditions. The
center of mass of an animal within the field of view is tracked and analyzed on a frame-by-frame
basis. Distance the animal travels and its time spent in particular ROIs are calculated. Software
and running the code generates plots which present the distance traveled. Method for tracking are
based on (Pennington et al., 2019).
Living Software Imaging Analysis
Images were analyzed using LIVING IMAGE software version 3.2 (Perkin Elmer).
Analysis was performed by measurement of total photon flux. The region of interest (ROI)
10
was manually selected over relevant regions of signal intensity. The area of ROI was kept
constant within experiments; dorsal ROI was 2 cm
2
, ventral ROI was 2 cm
2
, tissue ROI was 1
cm
2
, and Brain to tumor ratio ROI was 0.2 cm
2
. The intensity was recorded for each ROI, in
units’ total flux (photons/s/cm
2
/steradian).
Tissues
Animals were euthanized, necropsy performed and tissue samples were kept. Tumors
were dissected and weighted when mice were euthanized. The following organs were collected
for each experiment: Brain, Heart, Liver, Lungs, Adipose, Spleen, Bone, Skin, Blood, Prostate,
Kidney, Eyes, Stomach, Small Intestine, Large Intestine, Testes, Muscle, Bladder, and Seminal
Vesicles. Organs and tumors were removed and imaged to assess uptake and retention of NMI.
Tissues were snap-frozen and stored at -80°C.
Statistical Analysis
Statistical analysis was determined using GraphPad Prism 8 (GraphPad, Inc, USA).
Statistical significance was evaluated using the One-way ANOVA test for all in vitro
experiments; P < 0.05 was considered significant. One-way ANOVA test was used to compare
all treatment groups.
11
3.Results
The purpose of this in vitro study was to assess if NMI and TMZ are effective reducing
colony formation in GL26 cell line. Colony formation assay is an in vitro cell survival assay
based on the ability of a single cell to grow into a colony. The assay essentially tests every cell in
the population for its ability to undergo “unlimited” division. This assay is the method used to
determine cell reproductive death after treatment with the cytotoxic agent. After 14-day study
colonies were imaged to visually compare the three treatment groups: control, 5 µM NMI, or 45
µM TMZ. Based on Figure 3A it can be seen that NMI greatly inhibits colony formation
compared to TMZ. To further support this finding, the colony formations were quantified based
on percent area covering each well then graphed in Figure 3B. Control wells were considered
100%. When looking at the data NMI reduced colony formation with a mean of 24%, whereas
TMZ reduced colony formation with a mean of 62%. After conducting a one-way ANOVA test it
was shown that when comparing the control to NMI it was statistically significant (p<0.0001). It
was also shown that NMI reduction of colony formation compared to TMZ was statistically
significant (p<0.0009). NMI is potent inhibitor of colony formation a dose of 5 µM compared to
the current chemotherapy option TMZ at a dose of 45 µM.
12
Fig.3 NMI treatment reduces clonogenicity and growth of GL26. A) GL26 cells were treated NMI 5 µM or
TMZ 45 µM for 48 hours, then incubated another 10 days for the colony formation assay. Colonies were stained and
quantified. B) Control treated cells were considered to be 100%. NMI increased cytoxicity compared to control or
TMZ treated cells.
After conducting in vitro studies, it was discovered that NMI is a potential inhibitor of
glioma. It was decided to move forward and begin the in vivo studies. The studies conducted can
be seen in Table 1. The first study involved nontumor bearing mice. The purpose was to assess
the biodistribution of NMI. We wanted to figure out what route of administration would be used
for future studies. Intravenous administration is the standard used in majority of in vivo
experiments; oral administration was chosen because previous studies of NMI only used
intravenous injection. Testing oral administration was conducted to assess the clinical utility of
NMI the enteral route improves patient compliance. The second study extended the first study
with non-tumor mice by assessing uptake and biodistribution of NMI over 24h GL26 tumor
bearing mice and further studies were conducted using the xenograft mouse model. Intracranial
implantation of GL26 was conducted in order to effectively mimic clinical studies of glioma.
The findings from the first and second study were used to determine the route of administration
of NMI for further in vivo studies. The third study was to assess a single dose of NMI given to
13
tumor bearing mice, in order to determine if NMI was preferentially delivered to tumor tissue
compared to adjacent brain tissue. Biodistribution of NMI was assessed over a 1-week period to
further understand peak accumulation and signs of clearance after 1-week post treatment. The
final xenograft study was conducted to determine a dose response and optimal biodistribution
parameters for NMI. Results for each study are further discussed relative to each representative
figure below.
Table 1: In vivo studies performed with NMI treatment on glioma. Type of study, length of study, dosing of NMI,
administration of NMI, imaging time points
The first study was conducted to determine NMI pharmacokinetics and biodistribution in
nontumor bearing mice with Sulfobutylether-beta-cyclodextrin (SBECD) formulation. A single
dose of NMI 5 mg/kg was given either oral or intravenously. After the injection was given mice
were imaged at 1, 3, 10,15, 30, and 45 minutes. Imaging continued at 1, 4, and 24 hours after
dose was given. Figure 4A shows the real time distribution of NMI over the course of the 24-
hour period after the injection was given. NMI given intravenously was distributed rapidly as can
be seen on Figure 4A from when the first image was taken 1 minute after the injection and still
readily distributed 24 hours later post injection. As for NMI given orally it can be seen that it
was about 30 minutes until it showed it had been distributed in the mice. However, when looking
at the total flux in Figure 4B it can be seen that even though 5 mg/kg of NMI was orally
delivered, based on total flux not all of it was distributed compared to intravenous. This was
14
further supported when ex vivo imaging was done. Ex vivo was conducted to compare the
biodistribution throughout various organs. As can be seen in Figure 4C for NMI given
intravenously the majority of organs florescence indicating NMI was present in that organ. One
notable organ that showed no NMI detection was the seminal vesicles which are part of the male
reproductive system. When looking at the distribution of NMI given orally it was shown that
NMI was distributed in the lungs, kidneys, stomach, and large intestine. The stomach being the
highest fluorescence of NMI, followed by the lungs, large intestine, and kidney. It was noted that
no detection of NMI was in the small intestine, further studies are recommended to understand
the reasoning but it could be due to changes in pH throughout the Gastrointestinal Tract. The
fluorescence intensities were quantified in Figure 4D for each organ to better visually compare
the distribution of NMI in both delivery systems. After 24 hours it was shown that intravenously
given NMI reaches most organs including the brain, oral had minimal absorption except in the
organs mentioned earlier. Even though not show here, a second study was conducted using
wildtype GL26 tumor bearing mice. A single dose of NMI 25 mg/kg was given oral or 5 mg/kg
intravenously; after 24 hours it showed similar results to the first study. Mice were imaged and
ex-vivo imaging of tissues was done on the same day. One notable difference was in the brain
where it was seen that NMI preferentially accumulated in the tumor tissue and not in the
surrounding brain tissue. Based off of the results from the first two studies it was concluded that
intravenous administration would be used for the rest of the studies.
15
Fig 4. Biodistribution of NMI in C57BL wildtype mice. A) In vivo fluorescence 24 h imaging after
intravenous injection or oral gavage of NMI (5 mg/kg). B) Quantification of fluorescence of 24 h imaging was
plotted as total flux. C) Ex vivo imaging of organs for IV or oral administration. D) Quantification of fluorescence of
organs was plotted as total flux.
Study three was to determine distribution of NMI in one week in xenograft glioma
models focusing on the brain and tumor. A single dose of NMI 5 mg/kg was administrated
intravenously, in 4 different treatments groups, treatment groups are as followed: 24, 48, 72 hour
and 1 week prior to imaging day. Mice were imaged and ex-vivo imaging of all organs was done
same day. Figure 5A shows NMI distribution and accumulation in the tumor relative to the
surround brain tissue. Maximum accumulation was found at 48 hours after injection, and lasted
up to 7 days. By the 7 days after injection accumulation has weaken, however still present in
tumor. It must be noted that after 48 hours no accumulation is present in blood. One notable
result was that NMI distributed twice as much in the tumor compared to the surround tissue in
the brain.
16
Fig 5. Specific uptake of NMI by tumor but not by normal tissue. A) Preferential uptake and retention of 5
mg/kg NMI in mice tumor xenograft over a 7-day period. B) Analysis of ex vivo NMI accumulation shown as brain
to tumor.
The final xenograft study was done to see if there can be a dose response for NMI. Mice
were imaged and ex-vivo imaging of tissues was done same day. The goal was to confirm that
NMI targets glioma. This was done by using GL26 luciferase labeled cell line. After intracranial
implantation mice were imaged on day 13 of the study. Bioluminescence imaging was done to
conform that tumors were present in mice as seen in Figure 6A. This was done by giving an
intraperitoneal injection of D-Luciferin, waiting approximately 10 minutes to ensure entire
systemic circulation. Once images were captured, mice were treated with NMI. The treatment
groups were as followed: control, 0.5 mg/kg NMI, 5 mg/kg NMI, or 50 mg/kg NMI. The 5
mg/kg dose is the animal representative dose that would be translated into clinical studies, as for
the 0.5 mg/kg and 50 mg/kg doses those were chose to represent a dose 10 times less than the
clinical dose and 10 time greater than the clinical dose, in order to accurately test a dose
response. The treatments were given 48 hours prior to imaging. When imaging was conducted on
day 15, Figure 6B shows a dose response of NMI at the injection site on the tail, as well as on the
top of the head of each mouse. Mice were imaged and ex-vivo imaging of all organs was done
same day. The brain/tumor, liver, kidneys, heart, blood, and lungs were imaged on the same plate
as seen on figure 6C. For 0.5 mg/kg dose it was shown that after 48 hours NMI was detected in
17
the lung. For the 5 mg/kg NMI was accumulated in the tumor, as for the rest of the tissue the
relative amounts of NMI were consistent with findings from previous studies. For the 50 mg/kg
dose of NMI it can be seen that after 48 hours the organs are oversaturated with NMI which is
expected since it is representative of 10 times greater than the clinical dose of 5 mg/kg. After
organs were imaged the total flux was quantified and graphed Figure 6D. When examining the
data, the graph shows a dose response for each organ and tissue. At the end of this study it was
concluded that NMI can be taken up by the brain tumors after effectively crossing the blood
brain barrier and blood tumor barrier.
Fig 6. NMI Specifically targets cancer cells and shows a dose response in mice and ex vivo. Animals were
implanted with luciferase labeled glioma. After 10 days, NMI was administered i.v. injection (0.5 mg/kg, 5 mg/kg,
or 50 mg/kg) prior to imaging then imaged for A) Bioluminescent B) Fluorescence. Bioluminescence indicated brain
tumor sites with luciferase; and fluorescence identified sites NMI localization. C) Ex vivo imaging for the following
tissues: Brain. Liver, Kidney, Heart, Blood, and Lungs. D) Graph showing the dose response for 48-hour treatment
of NMI related to organs in part C.
18
To evaluate the toxicity of NMI, we injected NMI or Vehicle into nontumor bearing mice
by tail vein intravenous injection. The toxicity study was done over the course of a week. After
mice were dosed with toxic levels of NMI, weight was monitored over the course of a week
following the injection of NMI. Food and physical changes were also monitored over the course
of the week. Mice were imaged on day 7 and ex-vivo imaging of tissues was done same day.
Weight was measured every day the same time, then graphed as a percentage of starting weight.
Then graphed on box and violin graph to understand the overall changes of weight. For the
control mice experienced fluctuations in weight but over the period of the 1 week there was no
net change of weight were mean weight change was 0%. For 50 mg/kg mice experienced some
weight loss at the beginning but as the week went on gained that weight back, after 1 week there
was no net change of weight were the mean weight change was 0%. For the 100 mg/kg mice
weight loss was show, some mice did gain weight as the week came to an end, but after 1 week
there was a net change in weight where the mean weight was -2% indicated an overall weight
loss of 2% of their original weight. Lastly 150 mg/kg mice showed weight loss throughout the
entire week, even though some mice gained weight it was still well below their original weight.
After the one week there was a net change in weight where the mean weight was -7% indicating
an overall weight loss of 7% of their original weight. It was concluded that toxic doses of NMI
cause weight loss in mice. As mentioned before food consumption was monitored and showed
initial decrease of appetite, but after day 3 appetite was back to normal. Only physical changes
were the weight change, no other physical indications of changes were shown.
19
Fig 7. Body weight as a percentage of mice dosed with toxic levels of NMI. Depicted are mean weights as
percentage of starting weight.
To evaluate the toxicity of NMI, we injected NMI into nontumor bearing mice by tail
vein intravenous injection. The toxicity study was done over the course of a week. After mice
were dosed with toxic levels of NMI, behavior was recorded for an hour after the treatment.
Behavior was recorded by setting up a mounted camera with an aerial view of the cages.
Underneath the cages were white poster board to help create a contrast between the mice and its
surrounds. Cages were emptied of bedding and potential objects that mice could hide by.
Recording sessions were done during day for optimal lightening conditions. After completion of
the 1-hour recording session, mice were transported back to animal facility at USC, and housed
under their normal conditions. Video analysis was first done by watching the record session, this
was done to note any behavior changes. All mice responded to normal startle cues, showed signs
of some vertical behavior, and exploration throughout the cage. It was noted that some mice
traveled more than others throughout the cage, whereas others traveled in a set area of the cage.
Once visual analysis was done, computational analysis was done using a coding software called
ezTrack. The coding software has the ability to conduct location tracking of animal across the
course of recorded session. This is done by defining the center of mass of an animal within the
20
field of view then is tracked and analyzed on a frame-by-frame basis of the entire recorded
session. Distance the animal travels and its time spent in particular ROIs are calculated. Software
and running the code generates plots which present the distance traveled over the course of the
recorded session, heat maps to indicate preferential areas the animal was exploring, and travel
map to see the path the animal explored throughout the session. Figure 8A shows a frame by
frame analysis of distance traveled for each treatment group. The distance traveled is recorded in
pixels. The treatment groups were the following: control, 50 mg/kg NMI, 100 mg/kg NMI, and
150 mg/kg NMI. The control showed typical exploration behavior, initially not traveling very
much but as time went on and the mice became more comfortable in the environment you see a
steady increase in exploration. The 50 mg/kg showed sporadic exploration, as you see from the
figure there were moments of lethargy with little exploration, followed by moments of increased
exploration in the area. The 100 mg/kg showed mild exploration at the beginning of the session
followed by minimal exploration. Even though overall exploration was low it must be mentioned
that upon evaluating the heat maps which shows preferential areas, this treatment group preferred
to only explore a small area of the cage rather than the entire cage. Lastly 150 mg/kg treatment
group had more sporadic phases of exploration compared to the 50 mg/kg group. Based off the
frame by frame analysis of the recorded session, we quantified total distance traveled in the
entire one-hour session as seen in Figure 8B. After conducting a one-way ANOVA test it was
shown that the differences were not statistically significant amongst the four treatment groups (p
> 0.05).
21
Fig 8. High doses of NMI has minimal effect on exploration in mice. A) showed a frame by frame distance
traveled during session when the animal was given high doses of NMI (50-150 mg/kg). B) Total distanced traveled
of each treatment group.
Gel electrophoresis was conducted in order to determine what proteins NMI binds to.
Samples were chosen from the fourth in vivo study. The purpose was to see if NMI is detectable
and if we see a dose response of the amount of NMI bound to the proteins. After running the gel,
the gel was captured by iBright Imaging System it was shown that NMI binds predominately to
two proteins, which are indicated by the two predominant bands shown in Figure 9A. To confirm
that it was NMI bound to proteins we imaged the same gel under fluorescence conditions using
the same instrument. From Figure 9B NMI was indicated by fluorescing in red. Again, you see
22
two prominent bands on the gel which NMI fluorescence. After capturing the images, we
quantified the two bands of interest, by measuring the bands density, this was done using the
image from Figure 9A in order to get a reading for each sample loaded on the gel. Figure 9C
shows the quantification of bands 1 and 2 for each sample. The sample order legend corresponds
with the order the samples were loaded on the gel. It must be noted that you see a dose response,
indicating that more NMI is present the more it binds to those proteins of interest. One more
notable observation is when comparing the 50 mg/kg brain and it correspond tumor sample band
2 is more prominent in the tumor sample versus the brain sample. This shows a preferential
accumulation of NMI to the protein related to band 2. The bands are currently being studied in
order to identify what proteins that NMI is binding to are.
Fig 9. Gel of NMI bound to proteins. A) Gel showing a dose response of NMI bound to two unidentified
proteins represented by band 1 and 2. B) Same gel under fluorescence filters to show NMI. C) Quantification of
bands 1 and 2 for each sample. Samples order legend correspond with order of sample loaded in each lane on gel.
23
4. Discussion and Conclusion
Based off of the findings from the in vitro studies, NMI is a potential inhibitor of glioma.
MTT assay is used to measure cytotoxicity. Glioma cells were tested under three different
treatment conditions: NMI alone, TMZ alone, or NMI and TMZ combination treatment. The
MTT assay was conducted on all three glioma cell lines: GL26, U251S, and U251R. TMZ alone
had a low cytotoxic effect on each cell line reducing the survival from 100% to about 70%. A
cytotoxic effect can be seen with NMI where survival was reduced from 100% to 55%. When
combination treatment of 5 µM of NMI was added to TMZ, cell proliferation was reduced
instantaneously from 100% to 55-37% depending on which cell line was examined. NMI shows
cytotoxic effect on glioma cell lines, the combination of NMI and TMZ have the potential to
have synergistic effect in clinical studies. Colony formation assay is an in vitro cell survival
assay to assess if NMI and TMZ are effective reducing colony formation in GL26 cell line. NMI
greatly inhibits colony formation compared to TMZ. NMI reduced colony formation with a mean
of 24%, whereas TMZ reduced colony formation with a mean of 62%. NMI is potent inhibitor
of colony formation a dose of 5 µM compared to the current chemotherapy option TMZ at a dose
of 45 µM.
The purpose of in vivo studies was to assess the biodistribution of NMI. Intravenous
administration should be done for future clinical studies. After 24 hours NMI reaches most
organs including the brain. NMI given intravenously was distributed rapidly and still readily
distributed 24 hours later, oral had minimal absorption. NMI given intravenously all majority of
organs florescence indicated NMI was present in that organ. Potential concerns for the high
accumulation in the lungs. Intracranial implantation of GL26 was conducted in order to
effectively mimic clinical studies of glioma. NMI can be taken up by the brain tumors after
24
effectively crossing the blood brain barrier and blood tumor barrier. NMI accumulates in the
tumor twice as much compared to the surround brain tissue. Maximum accumulation was found
at 48 hours after injection and lasted up to 7 days. NMI still is present in the tumor after a week
in the body. NMI follows a dose response of inhibition, however a dose of 10 times more or
greater of the clinical dose shows oversaturation. It was concluded that toxic doses of NMI cause
weight loss in mice. NMI at toxic doses show no behavioral effects, mice responded to normal
startle cues, vertical behavior, and exploration. NMI binds to two proteins. There is preferential
binding to one of the proteins in tumor tissue samples compared to brain tissue samples. This
could be related to overexpression.
Future work would be to 1) Further test NMI alone or in combination with TMZ on other
glioma cell lines to see if the studies show similar results. 2) Repeat the colony formation assay
on U251R and U251S glioma cell lines. 3)Work out potential formulation issues, to design NMI
as oral chemotherapy drug, and reassess its biodistribution to determine its bioavailability ensure
it can effectively cross the blood brain barrier. 4) Further studies are recommended to
understand the reasoning NMI does not biodistributed in the small intestine.5) In vivo
experiments were done using intracranial model, in order to closely resemble clinical situations,
the disadvantage of this model is short lifespan (less than 15 days) and not human cancer cell
line. Due to this it is recommend that future work focusing on the long term effects of NMI
using mice that receive the sub-cutaneous implanted glioma or a less aggressive slower growing
cell line (Banissi et al., 2009). 6) Conduct future xenograft studies with female mice to see if
there are potential gender differences. 7) Extend the single dose of NMI past 1-week period to
known its clearance length. Since patients received multiple courses of chemotherapy in clinical
treatments, it is recommended to conduct a repeated cycle of chemotherapy in order to determine
25
the effect of repeated dosing of NMI and its cumulative effects in the body. 8) Further research
on NMI and its high distribution in the lungs. 9) Toxicity studies need to be further evaluated to
see if effects are reversible or irreversible. Future toxicity test should be extended past a week to
confirm if weight can be gained fully back to original body weight. Chronic toxicity studies need
to be conducted to see potential long-term effects of NMI. 10) Future behavior studies should
monitor exploration multiple times throughout the duration of the entire study. See if the
identified proteins NMI binds to are consistent in other glioma cell lines or just for GL26 glioma
cell line. 11) As mentioned before OATPs play a role in the delivery of NMI, as of now the
mechanism of action is unknown, so further studies are recommend to study the mechanism of
action involving the OATPs and NMI(Shi et al., 2014). In summary NMI can be used a
theranostic agent for brain cancer. NMI shows the capabilities to reduce tumor cell growth and
tumor cell survival during in vitro experiments. NMI is readily biodistributed when given
intravenous, it has preferential accumulation in tumor tissue, compared to the adjacent brain
tissue. The studies conducted in this manuscript contribute to the scientific literature by
showing the potentials of theranostic agents in treatment in glioma, as well as its potential to be
used in other cancer forms.
26
References
Aston, W.J., Hope, D.E., Nowak, A.K., Robinson, B.W., Lake, R.A., and Lesterhuis, W.J.
(2017). A systematic investigation of the maximum tolerated dose of cytotoxic chemotherapy
with and without supportive care in mice. BMC Cancer 17, 684.
Ausman, J.I., Shapiro, W.R., and Rall, D.P. (1970). Studies on the chemotherapy of experimental
brain tumors: development of an experimental model. Cancer Res 30, 2394-2400.
Banissi, C., Ghiringhelli, F., Chen, L., and Carpentier, A.F. (2009). Treg depletion with a low-
dose metronomic temozolomide regimen in a rat glioma model. Cancer Immunol Immunother
58, 1627-1634.
Caja, L., and Tan, E.-J. (2019). Epithelium to Mesenchyme Transition, 3rd edn (Academic
Press).
Cho, H.Y., Wang, W., Jhaveri, N., Torres, S., Tseng, J., Leong, M.N., Lee, D.J., Goldkorn, A.,
Xu, T., Petasis, N.A., et al. (2012). Perillyl alcohol for the treatment of temozolomide-resistant
gliomas. Mol Cancer Ther 11, 2462-2472.
D'Alessandro, G., Grimaldi, A., Chece, G., Porzia, A., Esposito, V., Santoro, A., Salvati, M.,
Mainiero, F., Ragozzino, D., Di Angelantonio, S., et al. (2016). KCa3.1 channel inhibition
sensitizes malignant gliomas to temozolomide treatment. Oncotarget 7, 30781-30796.
Ferrer-Font, L., Arias-Ramos, N., Lope-Piedrafita, S., Julia-Sape, M., Pumarola, M., Arus, C.,
and Candiota, A.P. (2017). Metronomic treatment in immunocompetent preclinical GL261
glioblastoma: effects of cyclophosphamide and temozolomide. NMR Biomed 30.
Fishburn, A.G. (1965). An introduction to pharmaceutical formulation. In The Commonwealth
and international library of science, technology, engineering and liberal studies Pharmacy and
pharmaceutical chemistry (Oxford, New York,: Pergamon Press), pp. 1-14.
Gao, J.Q., Lv, Q., Li, L.M., Tang, X.J., Li, F.Z., Hu, Y.L., and Han, M. (2013). Glioma targeting
and blood-brain barrier penetration by dual-targeting doxorubincin liposomes. Biomaterials 34,
5628-5639.
Kushal, S., Wang, W., Vaikari, V.P., Kota, R., Chen, K., Yeh, T.S., Jhaveri, N., Groshen, S.L.,
Olenyuk, B.Z., Chen, T.C., et al. (2016). Monoamine oxidase A (MAO A) inhibitors decrease
glioma progression. Oncotarget 7, 13842-13853.
Pennington, Z.T., Dong, Z., Feng, Y., Vetere, L.M., Page-Harley, L., Shuman, T., and Cai, D.J.
(2019). ezTrack: An open-source video analysis pipeline for the investigation of animal
behavior. Sci Rep 9, 19979.
Shi, C., Wu, J.B., Chu, G.C., Li, Q., Wang, R., Zhang, C., Zhang, Y., Kim, H.L., Wang, J., Zhau,
H.E., et al. (2014). Heptamethine carbocyanine dye-mediated near-infrared imaging of canine
and human cancers through the HIF-1alpha/OATPs signaling axis. Oncotarget 5, 10114-10126.
27
Shih, J.C. (2018). Monoamine oxidase isoenzymes: genes, functions and targets for behavior and
cancer therapy. J Neural Transm (Vienna) 125, 1553-1566.
Sugiura, K. (1969). Effect of L-asparaginase (NSC-109,229) on transplantable and spontaneous
tumors from mice and rats. Cancer Chemother Rep 53, 189-194.
Wu, J.B., Lin, T.P., Gallagher, J.D., Kushal, S., Chung, L.W., Zhau, H.E., Olenyuk, B.Z., and
Shih, J.C. (2015a). Monoamine oxidase A inhibitor-near-infrared dye conjugate reduces prostate
tumor growth. J Am Chem Soc 137, 2366-2374.
Wu, J.B., Shi, C., Chu, G.C., Xu, Q., Zhang, Y., Li, Q., Yu, J.S., Zhau, H.E., and Chung, L.W.
(2015b). Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and
targeted drug delivery for human brain tumor. Biomaterials 67, 1-10.
Yang, X.G., Mou, Y.H., Wang, Y.J., Wang, J., Li, Y.Y., Kong, R.H., Ding, M., Wang, D., and
Guo, C. (2019). Design, Synthesis, and Evaluation of Monoamine Oxidase A Inhibitors(-
)Indocyanine Dyes Conjugates as Targeted Antitumor Agents. Molecules 24.
Zhang, C., Long, L., and Shi, C. (2018). Mitochondria-Targeting IR-780 Dye and Its
Derivatives: Synthesis, Mechanisms of Action, and Theranostic Applications. Advanced
Therapeutics 1, 1800069.
Abstract (if available)
Abstract
Brain cancer is universal, it can affect all people regardless of age, gender, or ethnicity. Susceptibility for specific types of brain cancer varies. Glioblastoma multiforme (GBM) can be deadly where the average survival time is 14-15 months from the time of diagnosis. Previous work from this laboratory has shown that Near infrared dye MHI-148 conjugated Monoamine oxidase Inhibitor clorgyline (NMI) is a therapy and diagnostic (theranostic), therefore it can both treat and monitor the brain tumors. This is due to the targeting mechanism of NMI interacting with a specific transporter in cancer cells. Using in vitro and in vivo brain tumor models have shown of NMI to able to inhibit the development of glioma. NMI can be used as a theranostic for glioma. This study shows that biodistribution, route of administration and the toxicity level of NMI in the present and absence of brain tumor GL26 cells. When testing route of administration both in non-tumor and tumor bearing mouse model, it was found that NMI is readily distributed when administered intravenously, in comparison oral route of administration had minimal distribution in the body indicating intravenous administration of NMI was to be used for optimal biodistribution. Further biodistribution studies were conducted where the focus was on tumor bearing mouse model. In a one-week study it was learned that NMI accumulates in the tumor and not in the adjacent brain, peak accumulation at of NMI is 48h in tissue, however complete clearance of NMI in blood is 48h, and elimination from tissue was more than 1 week. In addition to NMI accumulation in the tumor and not in the adjacent brain, NMI follows the dose-response relationship in that as NMI dose increases accumulation occurs, but at 50 mg/kg it reaches maximum accumulation and is over saturated. Toxicology studies showed that at toxic levels of NMI caused weight loss, but minimal effect on food consumption or exploration. In summary, this study shows preferred route of administration, the safe and toxic dosages of NMI for the treatment and diagnosis of glioma. This new information provides important preliminary data for future pharmacokinetic and other preclinical studies.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Study of a novel near-infrared conjugated MAOA inhibitor, NMI, against CNS cancer by NCI60 data analysis
PDF
NMI: a near infrared conjugated MAO-A inhibitor as a novel targeted therapy for colorectal and other cancers
PDF
Bioinformatics analysis of the anti-cancer potency of NMI on non-small cell lung cancer and its potential mechanism
PDF
NMI (near-infrared dye conjugate MAO A inhibitor) outperformed FDA-approved prostate cancer drugs with a unique mechanism based on bioinformatic analysis of NCI60 screening data
PDF
Co-expression of monoamine oxidase A and prostate cancer stem cell markers in Pten knockout mice
PDF
Monoamine oxidase A inhibitors and androgen receptor antagonists regulate mitochondrial function in prostate cancer cells
PDF
MAO a deficient mice exhibit an altered immune system in the brain and prostate
PDF
Monoamine oxidase inhibitors regulate tumorigenesis and mitochondrial function in a prostate cancer mouse model
PDF
Potential therapeutic effect of monoamine oxidase (MAO) inhibitor on human neuroblastoma
PDF
Analysis of human brain endothelial cells versus human tumor associated brain endothelial cell tight junction and adherens junction expression differences in glioblastoma multiforme
PDF
Interleukin-11: a study of its effects in glioblastoma multiforme
PDF
Mechanistic studies of novel small molecule anti-cancer agents using next generation sequencing
PDF
Monoamine oxidase (MAO) knock-out mouse models: Tools for studying the molecular basis of aggression, anxiety, autism and cancers
PDF
Integrative mathematical and computational approaches to investigate metastatic cancers
PDF
Molecular targets for treatment of glioblastoma multiforme
PDF
Blockade of CXCR2 as a novel approach for cancer chemotherapy
PDF
Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme
PDF
Inhibition of MAO-A by Dual MAO-A/HDAC inhibitors: in silico approach for ligand binding and affinity prediction
PDF
Perimenopausal transition increases blood brain permeability: implications for neurodegenerative diseases
PDF
Inhibition of monoamine oxidase A and histone deacetylase inhibitors: computational prediction of ligand binding
Asset Metadata
Creator
Escobedo, Alesi Renee
(author)
Core Title
Assessment of theranostic agent for brain cancer
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Publication Date
04/30/2020
Defense Date
04/30/2020
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
brain cancer,GBM,glioblastoma multiforme,glioma,MAOA,monoamine oxidase A,NMI,OAI-PMH Harvest
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Shih, Jean C. (
committee chair
), Beringer, Paul M. (
committee member
), Duncan, Roger F. (
committee member
)
Creator Email
alesirenee@gmail.com,arescobe@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-292479
Unique identifier
UC11663930
Identifier
etd-EscobedoAl-8378.pdf (filename),usctheses-c89-292479 (legacy record id)
Legacy Identifier
etd-EscobedoAl-8378.pdf
Dmrecord
292479
Document Type
Thesis
Rights
Escobedo, Alesi Renee
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
brain cancer
GBM
glioblastoma multiforme
glioma
MAOA
monoamine oxidase A
NMI