“DESIGN AND CHARACTERIZATION OF PEPTIDE DECORATED NANOCARRIERS AS
CHEMOTHERAPUTIC AGENTS IN BREAST CANCER”
Ryan Lim
B.S., California State University, Sacramento, 2009
PROJECT
Submitted in partial satisfaction of
the requirements for the degree of
MASTERS OF ARTS
in
BIOLOGICAL SCIENCES
(Stem Cell)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011
“DESIGN AND CHARACTERIZATION OF PEPTIDE DECORATED NANOCARRIERS AS
CHEMOTHERAPUTIC AGENTS IN BREAST CANCER”
A Project
by
Ryan Lim
Approved by:
__________________________________, Committee Chair
Thomas R. Peavy, Ph.D.
__________________________________, Second Reader
Thomas Landerholm, Ph.D.
__________________________________, Third Reader
Jan Nolta, Ph.D.
____________________________
Date
ii
Student: Ryan Lim
I certify that this student has met the requirements for format contained in the University format
manual, and that this project is suitable for shelving in the Library and credit is to be awarded for
the project.
__________________________, Graduate Coordinator
Susanne Lindgren, Ph.D.
Department of Biological Sciences
iii
________________
Date
Abstract
of
“DESIGN AND CHARACTERIZATION OF PEPTIDE DECORATED NANOCARRIERS AS
CHEMOTHERAPUTIC AGENTS IN BREAST CANCER”
by
Ryan Lim
Breast cancer is the most common malignant tumor among women, accounting for an
estimated 24% of all cancer cases. Current therapies are clinically efficacious, but side effects
linked with these therapies are serious and can cause further harm not associated with the tumor
itself. It has been hypothesized that cancer stem cells allow for resistance to chemo- and radiation
therapy, and contribute to tumor spread (metastasis) and disease relapse. Therefore, the
development of a novel treatment method including the targeted delivery of cytotoxic agents to
the tumor mass for the treatment of advanced breast cancer is vital to improving the therapeutic
index and efficacy/toxicity balance.
Taking all of this into consideration, the goals of this project were to develop novel
strategies for discovering and using treatments against breast cancer. The use of a combination
therapy system for drug delivery should increase the therapeutic index, and help prevent the
relapse and metastasis of resistant/stem-like cancer cells. To accomplish these goals we used
several unique methodologies to discover and test new therapeutics. These methodologies
include: the One Bead One Compound (OBOC) screen for drug discovery, 3D culture tumor
modeling for drug screening, and nanoparticle systems for optimal drug-delivery.
The use of the One Bead One Compound (OBOC) combinatorial chemistry founded by
Dr. Lam and colleagues has allowed for the discovery of LMS peptides that bind specifically to
iv
breast cancer cells. These peptide ligands bind to high-mannose glycans specifically found on
breast adenocarcinomas. Screening these peptides using a 3D cell culture system has been shown
as an effective way to model the in vivo tumor response. The 3D culture system we use is an
efficient way to screen potential therapeutics, and has provided us data that suggests growth
inhibitory effects of the LMS peptides. This data suggests that the LMS peptides can be used as
both a targeting agent and therapeutic agent in our nanoparticle drug-delivery system. Our studies
show that functionalized super paramagnetic iron-oxide (SPIO) nanoparticles are a highly
efficient vehicle to use for this nanocarrier drug-delivery system. The LMS peptides,
nanoparticles, and chemotherapeutic drug, can all be used alone as therapeutic agents, but the
combined use of each of these agents will provide a treatment system that is much safer and
efficacious than conventional therapies.
Assessing the combined use of the LMS peptides and the nanocarrier system is the next
step in evaluating the ability of our combination therapeutic approach to increase therapeutic
efficacy. The results attained from this project have provided useful data that can contribute to
future discoveries in breast cancer treatment.
_______________________, Committee Chair
Thomas R. Peavy, Ph.D.
_______________________
Date
v
TABLE OF CONTENTS
Page
List of Figures ........................................................................................................................ vii
INTRODUCTION .................................................................................................................... 1
METHODS AND MATERIALS ............................................................................................ 14
RESULTS .............................................................................................................................. 22
DISCUSSION ........................................................................................................................ 39
LITERATURE CITED ........................................................................................................... 45
vi
LIST OF FIGURES
Page
Figure 1. Illustration of the tumor microenvironment .................................................................... 5
Figure 2. Illustration of 3D culture method and drug screening ...................................................... 8
Figure 3. Illustration of OBOC screening ...................................................................................... 12
Figure 4. OBOC library synthesis .................................................................................................. 16
Figure 5. Graphic representation of surface glycan composition .................................................. 23
Figure 6. OBOC screening of LMS peptides ................................................................................. 25
Figure 7. Mammospheres in 3D liquid suspension ........................................................................ 27
Figure 8. ZR-75-1 mammospheres in PVA hydrogel .................................................................... 29
Figure 9. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides at time zero ................. 31
Figure 10. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides after one week .......... 32
Figure 11. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides after three weeks ...... 33
Figure 12. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides after 4 weeks ............ 34
Figure 13. Nude female mouse with ZR-75-1 xenograft ............................................................... 36
Figure 14. Nude female mice with ZR-75-1 xenograft, and SPIO biodistribution ........................ 38
vii
1
INTRODUCTION
Breast cancer is the most common malignant tumor among women, making up 24% of all
cancer cases (1). It is a major health problem not only in the United States, but also individuals
from all socio-economic groups throughout the world are affected. Despite the intensity of
treatment with surgery, chemotherapy, and radiation therapy, breast cancer remains the second
most lethal cancer for women, accounting for 18% of all cancer related deaths (1). This is due to
many factors which will be discussed in the upcoming paragraphs that make not only most breast
tumors very aggressive, but allow for the tumor to metastasize before clinical detection and
treatment. It is because the quality of life for many individuals has been greatly affected that new
novel treatments must be developed.
Breast cancer can be subdivided into different subsets that have different molecular
pathologies and clinical significance. Although many subdivisions have been well established,
therapies for each type has not been. Subtypes have been defined as Human Epidermal growth
factor receptor 2 (HER2) positive, Estrogen receptor (ER) and Progesterone (PR) positive, as
well as Mucin (MUC) positive, and triple negative (negative for HER2, ER and PR). Each
subtype contributes to breast cancer progression by way of different growth factor signal
transduction pathways. In many cases, major proteins involved in the signal transduction pathway
that contribute to the pathology will dimerize (bind together in pairs) and cause signaling without
the need for stimulation by external growth factors. In addition, overexpression of these proteins
allows for increased signaling.
The aggressive nature of the cancer and ability to resist treatment is greatly affected by the
specific subtype. Each subtype has a unique pathway that if unregulated attributes to the
2
progression of the cancer, driving normal cells to overexpress key genes that will contribute to the
“six hallmarks of cancer”. Weinberg et al. (2000) defined the six “hallmarks of cancer” as 1)
acquisition of self-sufficient pro-growth signals, 2) insensitivity to anti-growth signals, 3)
apoptosis evasion, 4) immortality, 5) angiogenesis, and 6) invasion and metastasis to other tissues
(2). These are the standards required for normal cells to become malignant and cancerous.
But, within each of these fields there are no preset gene expression patterns which can easily
be defined and targeted. In cancer cells, key genes and proteins that control the cell cycle or the
ability to proliferate need to be altered from their normal function such as the protein
retinoblastoma, a major tumor suppressor protein that keeps cells from uncontrolled proliferation.
Breast cancer subtypes are defined by the major proteins that contribute to the progression of the
cancer, and each sub-type can be controlled by numerous genetic abnormalities that are quite
different. Although the establishment of these subtypes has allowed researchers to develop
treatments that specifically target them and help destroy the tumor, there are still a high number
of cases where the tumor does not respond or relapses occur. This is why new and novel
therapeutics need to be discovered for all sub-types of cancer so that they can be used in the
clinical setting.
The most promising targets for the development of novel therapeutics are the key proteins
involved in cancer progression. One specific target that has recently been shown to contribute to
the most aggressive tumors is the presence of high-mannose glycans found on the surface of the
cell. Research from Dr. Lam’s laboratory has shown that the more aggressive subtypes highly
express these surface glycans including: HER2 positive, chemo-resistant, and MUC positive.
Surface glycans have an important role in tumor progression; not only contributing to the six
hallmarks of cancer, but allowing for “stemness” (3). “Stemness” is the ability of an adult cell to
3
act and have features that are only found in stem cells. Cancer cells have stem cell-like features
since they have attained the ability to self-renew, proliferate rampantly, and possibly have multicellular differentiation potential. Cancer cells of large aggressive tumors need to gain stem-like
properties in order to survive and proliferate. The acquisition of these stem-like properties allows
for the cells to adapt to the changing environment of the tumor and allows for the following: high
levels of proliferation, unlimited self-renewal, drug resistance, and differentiation capacity.
Overexpression of surface glycans has been shown to contribute to proliferation,
invasion, angiogenesis, and dissemination through the circulatory system which are all attributes
required for the progression and metastasis of tumors (3). Glycoproteins are proteins which have
been glycosylated post-translationally by an addition of a glycan or polysaccharide. They have
many roles and responsibilities in a normal cell such as facilitating protein folding, allowing
proper protein function, coating of the cell surface for protection, and participating in cell-to-cell
or cell-to-extracellular matrix (ECM) interactions. When analyzing the surface expression of
glycans in different breast cancer cell lines and comparing those with normal breast tissue and
human embryonic stem cells (hESCs), high levels of surface glycans were found to be expressed
on hESCs as well as the more aggressive cancers (4,5). These breast cancer cells express very
similar levels of surface glycans as hESCs which are much higher compared to normal breast
cells. Specifically, high-mannose glycans are being found in high amounts on surface
glycoproteins and seem to be replacing the normal complex/hybrid glycans found on the normal
cell surface (4,5). Stem-like cancer cells are hypothesized to be the cause of tumor aggression,
metastasis, and relapse. Since these unique cells have been shown to overexpress surface glycans,
much like hESCs, it is more than apparent that we can take advantage of this fact and try to treat
the tumor with therapies that directly target these glycans.
4
The tumor microenvironment is another important factor which not only affects the
progression of the tumor and cancer cell “stemness” but also the way we can mimic in vivo tumor
growth and treatment. The tumor microenvironment is composed of about four different layers:
the acidic front, the vascularized circumference, the hypoxic region, and the necrotic core (Figure
1). The acidic front is the zone directly surrounding the tumor mass, which has low pH levels due
to the high levels of proliferation on the outer surface of the tumor. This high level of
proliferation causes increased metabolic waste which creates this acidic zone. The vascularized
circumference is the region of high proliferation just inside the acidic front which is where the
tumor is rapidly dividing and in need of additional nutrients. Thus, these proliferating cancer
cells express signaling proteins for angiogenesis to stimulate the growth of new blood vessels into
the region which produces a leaky vasculature that has penetrated the tumor mass. Since the
vasculature penetrates only the surface of the tumor, the newly recruited blood vessels are able to
carry oxygen and nutrients into the tumor mass only to these highly proliferative cells at the
surface. Just inside this region is the hypoxic region, containing low levels of nutrients and
oxygen, and comprises the bulk of the tumor. The center of the tumor contains the necrotic core, a
region containing many dead cancer cells that have died due to lack of nutrients and oxygen. This
region is also responsible for the selection of treatment resistant cancer cells and cancer stem cells
because of its microenvironment. It is because of the tumor microenvironment and the
development of a heterogeneic tumor that today’s therapeutics do not fully treat the patient.
Limited penetration of the therapeutic agent allows for the more aggressive resistant/cancer stem
cells to repopulate and cause a relapse. Only very resilient cells can survive in the necrotic core,
which puts a selective pressure on cells with stem-like properties to survive this harsh
environment.
5
Figure 1. Illustration of the tumor microenvironment. The tumor microenvironment is
composed of several specific regions including: The acidic front, highly proliferative
vascular region, the hypoxic region not penetrated by the vasculature, and the necrotic core
which selects for resistant cancer cells and cancer stem cells.
6
Since cancer stem cells seem to have such an important role in cancer progression, many
investigators have been trying to identify their origins and properties. Two models of tumor
progression have been proposed and cancer stem cells are central to both. The clonal expansion
model selects for clonal populations that have acquired genetic mutations over generations to
become cancerous and stem-like. Successive generations of a single cell and all of its progeny
acquire genomic mutations that provide selective growth and survival advantages to the daughter
cells. Daughter cells from later generations eventually accumulate key mutations that give it
stem-like properties. Alternatively, the hierarchal model proposes that a stem cell or mother cell
acquired all the genetic mutations to become cancerous (instead of progressively), which then
produces progeny that amplify this population of cancerous cells thereby forming the tumor. Both
models reveal the limitations that traditional therapies have when trying to attack a heterogeneous
tumor population that contains multiple cell types within. In both models, the unique stem-like
properties of a subset of these cancer cells allows for resistance to therapeutics and relapse.
Due to the tumor microenvironment, heterogeneity, and genomic instability, single targeted
therapies (e.g. chemotherapeutic agents) often result in the development of resistance. Thus, it is
essential that effective combination therapies be developed to rid the patient of cancer.
Combination therapy uses a combination of: general/traditional chemo- and radiation
therapeutics, specifically targeted therapeutics, and therapeutics that alter gene expression.
Traditional therapeutics generally work by killing actively dividing cells which will effectively
target cancer cells since they lack control of their cell cycle control and proliferate quickly. This
is an effective approach but will also cause damage to normal cells since there are a variety of
normally proliferating cells. This is why chemo- and radiation therapeutics result in hairloss and
loss of appetite, among other side effects. However, this traditional method of treatment can still
7
be useful in combination therapy in order to kill the bulk of the tumor mass by, non-specifically
killing all highly proliferative cells.
Other cancer treatment approaches involve the use of specifically targeted therapies which
includes small molecule drugs, antibodies, and synthetic peptides. These target very specific
proteins that contribute to the cancer progression, such as Herceptin which targets Human
Epidermal growth factor Receptor 2/neural tumor gene (HER2/neu) positive cancer cells. These
therapies often directly target the defining feature of the cancer subtype which is the major
pathological protein that contributes to tumor progression. Another novel therapeutic approach is
to alter the gene expression of the cancer cell by activating or inhibiting a specific protein that
controls gene expression. This alteration of gene expression can help steer the cancer cells away
from resistance and “stemness” allowing them to succumb to the other therapeutics and death.
In order to add more treatment options, novel screening techniques need to be implemented to
mimic the tumor in its natural state and determine the effectiveness of potential therapeutics
before moving to in vivo testing. It is quite apparent that cells cultured three dimensionally (3D
culture) would respond differently to therapeutics as compared to cells in a traditional two
dimensional space, such as cells grown on a flask surface. For one, the therapeutic agent would
not need to penetrate a tumor mass when cells are grown in a culture flask since they form a
monolayer. But, there are many other differences which make 3D modeling a better in vitro
study (6). Cellular morphological changes, signaling, and cell to cell interactions are all aspects of
3D modeling that are not seen in the normal 2D cultures. Figure 2 shows a graphical
representation of cells in a 3D culture. The three dimensional environment allows for cells to
grow in a more natural state which includes cell-to-cell and cell-to- ECM interactions in a three
8
Figure 2. Illustration of 3D culture method and drug screening. Cancer cells form
spheroid colonies in 3D culture hydrogels. The components that make up the hydrogel
are biotinylated poly vinyl alcohol (PVA) and polyethylene glycol (PEG) boronic acid
crosslinkers.
9
dimensional space similar to in vivo conditions. Cell surface receptor and overall gene expression
is altered due to this difference in spatial organization between 2D and 3D culture systems. Cell
morphological and polarization changes can be seen in 3D cultures that are not seen in 2D
culture. These changes are clearly controlled by the spatial organization of the cells interacting.
Cells within the center of growing colonies will respond and signal differently as compared to
cells on the outer layers creating differences in gene expression profiles of cells within a single
colony. Therefore, 3D cultures are a better model to mimic the tumor microenvironment during
therapeutic screening and will help us better understand the tumor response to therapeutics (6).
A promising future therapeutic approach involves the field of nano-medicine, a high-tech and
newly expanding field of medicine that uses nano-sized particles to help diagnose or treat a
diseased patient. Nanotechnology is an emerging field that has shown promise for the
development of novel diagnostic, imaging and therapeutic agents for a variety of diseases,
including cancer(7). Nanocarrier systems have been developed to deliver toxic chemotherapeutics
agents which limits the side effects normally seen in traditional therapies (7,8). The types of
nanocarriers used include solid nanoparticles, (9,10) liposomes, liposome-nanogel, dendrimers,
polymeric micelles and water soluble polymers (7,8) all of which are considered non-toxic. As
mentioned, traditional chemotherapeutics are limited by their general cytotoxicity and systemic
spread which include neurotoxicity (paclitaxel, vincristine, and vinblastine), cardiotoxicity
(doxorubicin), pulmonary toxicity (bleomycin), renal toxicity (cisplatin), among other side
effects. In addition, the therapeutic index is low in most cases, meaning cancer cell death is not
high enough before associated side effects are experienced. Thus, this limits the dose of the
therapeutic agent the clinician can administer which can result in a relapse if the entire tumor is
not eliminated.
10
It is believed that nano-formulations will be able to decrease the toxicity to normal organs,
increase the circulation time, slow down the metabolism of the drug, and facilitate the delivery of
the drug to the tumor sites due to an enhanced permeability retention effect (7,8). Most
nanocarrier systems conceal the drug from the immune system and they themselves are nonimmunogenic. In addition, packing the drug into the nanoparticles can decrease the metabolism.
Both of these attributes will allow for greater circulation time and thereby allow more of the drug
to accumulate at the tumor site. Enhanced retention of nanocarriers at tumor sites has also been
observed which appears to be due to the leaky vasculature found penetrating the tumor. The leaky
vasculature that is created by the tumor’s signal for angiogenesis floods the tumor with blood.
This increased vasodilation and blood flow helps the nanoparticles pour into the tumor site and
retains them there until they are degraded which then releases the drug. Superparamagnetic iron
oxide nanoparticles have been used for delivering drugs and for imaging the localization of these
particles to further understand this phenomenon. Drugs and targeting ligands can be conjugated
onto the surface of these iron oxide nanoparticles. This allows for increases in tumor targeting,
uptake, and therapeutic index of conjugated drugs. In addition to being able to determine the
location of the particles by magnetic resonance imaging, these particles can be heated up (i.e. site
specific heat induction) which contributes to tumor death and adds another combinational therapy
to the arsenal.
With the use of nanomedicine and a new method termed the One Bead One Compound
method (OBOC), it is possible to synthesize peptides and small molecules that can be conjugated
directly to nanoparticles to target and eliminate tumor cells. Using knowledge of traditional solidphase fmoc chemistry, Lam et al. (1991) founded The OBOC combinatorial chemistry method,
which allows for the high-throughput synthesis and screening of peptide and small molecule
11
chemical libraries using an on-bead screening assay(11). This novel method was founded by
combining traditional solid-phase fmoc chemistry on resins with the split-mix method. The splitmix method allows one to synthesize a large random library of peptides or small molecules onto
many different beads by conjugating a string of amino acids on the surface of the beads and
splitting/mixing the beads into different tubes of amino acids before each conjugation step. Each
resin bead will contain its own individual sequence which is the reason for the technique’s name,
“One Bead One Compound”. A large library of beads and compounds can then be screened
against one cancer type and individual beads of interest can be selected if found to cause cell
binding (Figure 3). The peptide on the bead can then be identified by having it sequenced. This
method can be used to discover small molecules of important biological relevance to cancer
treatment.
The OBOC technique has been modified to select for more than one cancer cell molecules
involved in cell signaling and pro-apoptotic ligands. The “One Bead Two Compound” (OBTC)
method can be used to detect a second cancer molecule by the following process: the first
compound that was detected by OBOC will be conjugated to beads and the bead further
processed by synthesizing and conjugating a new randomized small molecule onto individual
beads. Thus, each individual bead will have one known compound (the positively screened
compound) and a second randomized small molecule. The beads are then screened against a cell
line and immunocytochemistry is conducted to determine which of the compounds from the
random library can activate or inhibit specific proteins important in carcinogenesis. In addition, a
third cancer cell component can be identified by the “One Bead Three Compound” (OB3C)
12
Figure 3. Illustration of OBOC screening. One Bead One Compound (OBOC) screening of
a random synthetic peptide library. Unique compounds that interact with the cells will
cause the cells to bind to the beads as depicted above.
13
technique. The design is similar to the OBTC method in that a third randomized small molecule
will be conjugated to beads containing the other two compounds identified. This third random
library of peptides will be screened using immunocytochemistry to see if it can serve as an
antagonist against the signaling of the second compound (determined from OBTC). With these
techniques, it is expected that novel glycan binding peptides can be identified that target
aggressive breast cancer cells overexpressing high-mannose surface glycans. This will allow for
the selective targeting of tumor cells that are more stem-like. Targeting these stem-like cancer
cells for the delivery of high levels of therapeutics should be a more effective approach to treating
the resistive cells that allow cancer to persist.
The goal of this project is to use the OBOC method, nanotechnology, combination
therapy, and novel 3D culture screening models to improve the therapeutic index and
efficacy/toxicity balance when treating breast cancer patients. The OBOC method will be used to
screen many random peptide libraries in hopes of identifying unique peptides with biological
function that can bind breast cancer cells. Once a few potentially unique peptides have been
found, a novel method of 3D culture will be used to assess their functional ability to inhibit
cancer stem cells. Lastly, in vivo biodistribution and toxicity will be analyzed using doxyrubicin
loaded superparamagnetic iron oxide nanoparticles to assess visually the individual success of the
drug delivery system. The results from this initial in vivo assessment will be compared to
additional in vivo biodistribution studies but with drug-loaded particles decorated with the
aforementioned targeting peptides. These experiments will allow us to assess whether this novel
approach can be successfully used to target cancer stem cells and be used in future combination
therapies.
14
METHODS AND MATERIALS
Chemical reagents and resins
TentaGel S NH2 resin (90 μm, 0.26 mmol/g) was purchased from Rapp Polymere GmbH
(Tübingen, Germany). Rink amide MBHA resin (0.5 mmol/g), amino acid derivatives, HOBt, and
DIC were purchased from GL Biochem (Shanghai, China). All solvents and other chemical
reagents were purchased from Aldrich (Milwaukee, WI) and were analytical grade.
HPLC
Analytical HPLC was performed using a Waters 2996 HPLC system equipped with a
Waters Xterra® MS C18 column (4.6 × 150 mm; 5.0 μm), and employed a 20 min gradient from
100% aqueous H2O (0.1% TFA) to 100% CH3CN (0.1% TFA) at a flow rate of 1.0 mL/min.
Cell culture
WA01 Human embryonic stem (H1) cells were purchased from Wisconsin International
Stem Cell Bank (Wicell; Madison, WI, USA). H1 cells were cultured in K/O DMEM F-12
supplemented with KOSR, glutamine, NEAA, 2-mercaptoethanol, and b-FGF. Human breast
adenocarcinoma (MCF7) cells and Human breast ductal carcinoma (ZR-75-1) cells were
purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). MCF7 cells
were cultured in EMEM supplemented with FBS and NEAA. ZR-75-1 cells were cultured in
RPMI-1640 supplemented with FBS. Human breast adenocarcinoma (MCF7 HER2+, MCF7 C6)
and human breast immortalized normal (MCF10a) cells were gracious gifts from Dr. Jian Jian Li.
MCF7-HER2 and -C6 cells were cultured in EMEM supplemented with FBS, and NEAA.
15
MCF10a cells were cultured in MEBM supplemented with FBS, cholera toxin, insulin, and
NEAA. All cell lines were cultured as recommended by ATCC.
Synthesis of focused “one-bead one compound” peptide libraries
Fmoc chemistry1 and HOBt/DIC coupling were used to synthesize the peptides on
TentaGel S NH2 and Rink amide resin (loading 0.26 mmol/g and 0.5mmol/g, respectively). A
three-fold molar excess of Fmoc-protected amino acids to resin were used for coupling. Six-fold
molar excess of D-biotin to Rink Resin was used for coupling. The reaction was monitored with
the ninhydrin test, to detect the presence of a free amine group during coupling. The reaction was
blue after deprotection of fmoc from the amine and yellow when a new amino acid was coupled
(fmoc protects the amine group from reaction). The fmoc group was deprotected using 20%
piperidine in DMF. The peptides were finally cleaved from Rink resin using a TFA cocktail
containing 82.5% TFA: 5% phenol: 5% thioanisole: 5% H2O: 2.5% triisopropylsilane (TIS),
followed by precipitation in cold diethyl ether. The crude peptides were then purified by HPLC as
described above (Figure 4).
16
Figure 4. OBOC library synthesis. Schematic of solid-phase fmoc combinatorial chemistry
method to create OBOC libraries.
17
On-bead screening assay
H1, MCF 10a, MCF 7, MCF 7 HER2, MCF 7 C6, ZR-75-1 cells adherent to the bottom
of a T75 flask were trypsinized with 0.05% trypsin- EDTA and neutralized with culture medium.
Floating cells were collected, centrifuged, and resuspended in 10mL culture medium in a 10cm
Petri dish. The library beads were washed sequentially with ethanol, water, and PBS. The beads
were then incubated with suspended cells, and the entire dish was agitated at a speed of 40 rpm
inside an incubator at 37°C under 5% CO2. The plate was then examined using an inverted
microscope every 15 minutes for several hours to detect binding of cells to bead. A 24 hour time
point was analyzed as well.
Glycomics studies
Surface glycan distribution and content was analyzed by members of Dr. Carlito
Lebrilla’s laboratory (University of California, Davis). H1, MCF 10a, MCF 7, MCF 7 HER2,
MCF 7 C6, ZR-75-1 cells were lysed and glycoproteins were isolated and analyzed by mass
spectrometry to determine type and abundance. Cells membranes were extracted by
ultracentifugation. The membrane fraction was then taken and protein degradation conducted. Nlinked glycans were enzymatically release and ethanol precipitation was used to purify the glycan
fractions. Purification and enrichment of glycans was performed by using Graphitized CarbonSolid-phase extraction (SPE) (4,5). Then glycans were mass profiled for their mass and structures
elucidated by Matrix Assisted Laser Desorption/Ionization Fourier transform- ion cyclotron
resonance Mass spectrometry and tandem mass spectrometry (MALDI FT-ICR MS) (4,5).
Glycoside hydrolase studies were conducted to determine LMS peptide binding affinity.
18
Glycoside hydrolases were used to cleave surface glycans from cell lines and binding affinity was
tested using LMS peptides.
Synthesis of biotinylated PVA and gel cross-linker
Poly vinyl alcohol (PVA) was biotinylated using liquid-phase polymer chemistry
described in detail in the supplementary section. An amine group was coupled to the PVA using
sodium hydride 60% in mineral oil, epichlorohydrim, and ammonium hydroxide 30-40%. PVA
was dissolved in DMSO, and mixed with sodium hydride 60% dispersion in mineral oil for 2
hours. Epichlorohydrim was added and mixed overnight. Then ammonium Hydroxide 30-40%
was added and mix overnight. PVA was washed with ice cold ethanol to form a precipitate and
centrifuged. PVA was then washed with acetonitrile. The precipitate was dissolved in DMSO.
Then D-biotin was coupled overnight. Three-fold molar excess of biotin was used for coupling
biotin. Remaining amine groups were capped with acetic anhydride in the presence of N,NDiisopropylethylamine (DIEA). The product was precipitated in cold ethanol anhydrous and
purified through dialysis and lyophilized to concentrate the polymer. A 4-arm polyethylene glycol
(PEG) boronic acid cross-linker was synthesized using liquid-phase chemistry and
Hydroxybenzotriazole (HOBt)/ N,N′-Diisopropylcarbodiimide DIC/DIEA. Twelve-fold molar
excess of 4-carboxyphenyl boronic acid, pinacol ester was used for coupling on 4-arm PEG-NH2.
4 arm PEG-NH2, 4-carboxyphenyl boronic acid, pinacol ester, was dissolved with HoBt in DMF.
This solution was then mixed with DIC and DIEA overnight. The reaction was monitored with
the ninhydrin test. The product was finally cleaved using a Trifluoroacetic acid (TFA) cocktail
containing 50% TFA: 50% Dichloromethane (DCM), followed by precipitation in cold diethyl
ether, dialysis, and lyophilization.
19
Three dimensional cell culture and peptide screening
Mammosphere cultures were plated in 100mm ultra-low adherent culture dishes from
Greiner Bio-one (Monroe, NC, USA). Mammocult medium from Stem Cell Technologies
(Vancouver, BC, Canada) was used for liquid suspension 3D culture. Cells were cultured under
recommended conditions by Stem cell Technologies. Photographs were taken with the EVOS
phase-contrast microscope with built in digital camera at various time points to depict the growth
and proliferation of the spheroids. The size of the spheroids were measured to analyze growth and
proliferation. All 3D gel culture models were plated in 8-well chamber slides. Briefly,
biotinylated PVA was added to a mixture of cells (1x103), ddiH2O, 10% fructose, and 1µM
streptavidin conjugated to 1 µM biotinylated peptide (only streptavidin if control). Since both the
peptides and PVA were biotinylated, streptavidin served to bind the two together. PVA mixture
was added to 4-arm Boronic acid cross-linker to form a gel within wells. Combine biotinylated
PVA solution in medium with 1x103 cells, biotinylated peptide, 10% fructose solution in water,
streptavidin, and deionized water, allowing conjugation for 2 minutes. Add crosslinker to the
culture well. Layer cell solution onto crosslinker, and add additional crosslinker. Allow solution
to gel and add additional medium with 5% crosslinker to cover entire gel surface. Appropriate
ATCC recommended culture medium was added to the top of the gel. After two hours time,
media was changed and subsequently changed every 3 days for 5 weeks time (more details of the
3D culture method can be found in the supplementary section). Photographs were taken with an
EVOS phase-contrast microscope with built in digital camera at various time points to depict the
growth and proliferation of the spheroids. In vitro cytotoxicity was examined using a basic
propridium iodide (PI) staining protocol. In addition, cells were stained for apoptosis by adding
PI directly to the culture wells at 2µg/mL. The plate was then examined for orange 562-580nm
20
fluorescence under an inverted fluorescent microscope every 15 minutes for 2 hours after
excitation of PI at 488nm.
Xenograft
Female athymic nude mice (Nu/Nu strain), 6–8 weeks age, were purchased from Harlan
(Livermore, CA). All animals were kept under pathogen-free conditions according to AAALAC
guidelines and were allowed to acclimatize for at least 4 days prior to any experiments. All
animal experiments were performed in compliance with institutional guidelines and according to
protocol No. 06-12262 approved by the Animal Use and Care Administrative Advisory
Committee at the University of California, Davis. Animal studies were performed according to a
protocol approved by IACUC of the University of California, Davis. Female athymic nude mice
(nu/nu), obtained from Harlan (Indianapolis, IN) at 8–10 weeks of age, were injected
subcutaneously in the right lower flank with 10×106 ZR-75-1 cells suspended in 200 μL PBS.
When the subcutaneous tumors reached 0.5 to 1.0cm in diameter or 40–50 days after
implantation, the tumor-bearing mice were subjected to in vivo and ex vivo (necropsy) imaging
studies. Biodistribution of the nanoparticles were analyzed in a fluorescent imaging machine as
described in the next section.
In vivo screening and biodistribution
Doxyrubicin loaded Superparamagnetic Iron Oxide particles (SPIO) was prepared by
mixing 1:5 ratio overnight at 4°C, and subsequently injected into the tail vein, intraperitoneal, and
intratumoral regions in an anesthetized mouse before imaging. Animals were placed on a
transparent sheet in the supine position. Images were acquired with a Kodak IS2000MM Image
21
station (Rochester, NY) with a 625/20 band pass excitation filter, 700WA/35 band pass emission
filter, and 150 W quartz halogen lamp light source set to maximum. Data was collected at
different time points and analyzed using the Kodak ID 3.6 software by drawing the region of
interest (ROI) on the imaged mouse. For ex vivo imaging, the mice were euthanized and their
organs were excised for imaging.
22
RESULTS
Glycomics studies
Tandem MALDI FT-ICR MS studies were conducted by the members of Dr. Lebrilla’s
laboratory (UC Davis) to analyze the surface glycome content of several cell lines. These mass
spectrometry studies were conducted on several breast cancer cell lines, hESCs, and normal
breast cells; the cells lines included: H1, MCF7, MCF7 HER2, MCF7 C6, MCF10a, ZR-75-1,
and BT549 cells. The mass spectrometry results show unusually high expression of surface highmannose glycans in both hESCs and aggressive cancer lines. Figure 5 shows the glycome content
and percentage of several breast cancer cell lines as well as H1 hESCs and MCF10a normal
immortalized breast cells.
Analysis of H1 hESCs show extremely high levels of high-mannose glycan expression
(74%). The remaining 26% of surface glycans were comprised of other complex/hybrid glycans.
When comparing these results to adult normal cells not categorized as stem cells, the percentage
of complex/hybrid glycans was found to be much more abundant. MCF10a cells are immortalized
normal breast cells, and in this study they are used to represent the normal adult cell population.
These cells only contain 28% high-mannose glycans, whereas higher levels of complex/hybrid
glycans were found (72%). The analysis of different breast cancer cell lines provides an array of
differential glycan expression. Interestingly, the cancer cell lines believed to be more aggressive,
both HER2 positive and C6 chemo-resistant MCF7 cells, show similar levels of high-mannose
glycan expression to that of H1 hESCs. These cell lines show an expression level of surface highmannose glycans at 78% and 72%, respectively. The less aggressive MCF7 and ZR-75-1 cancer
cells have reduced expression of the high-mannose glycans (50%). Conversely, we did find
23
Figure 5. Graphic representation of surface glycan composition. Glycome profiles of H1
hESC, MCF10a normal breast tissue, and several breast cancer cell lines: MCF7, MCF7
HER2, MCF7 C6, ZR-75-1, BT549. Isolated and enriched glycans were analyzed by mass
spectrometry and tandem mass spectrometry to determine their mass and structures.
24
differential glycan expression profiles in BT549 cells as compared to the other cancer cell lines.
Although most cancer cells show higher expression of high-mannose glycans, these cells showed
a diminished level of expression consisting of only 7%, and an increase in complex/hybrid
glycans to 93%. The glycan profile of these cells still show aberrations in levels of expression
and content when compared to normal tissue, MCF10, since normal breast cells shows 28% highmannose glycan expression and BT549 cells have only 7% expression.
OBOC screening
OBOC screening was conducted using the LMS random peptide library. These beads
were screened against ZR-75-1 breast cancer cells, to assess for any binding interaction. Beads
were incubated with cells that were dissociated to create a single cell suspension in culture
medium. Cell binding to the beads was observed within 15 minutes. As seen in Figure 6, LMS3
(sKlPvA-EBES-K(Biotin)), LMS9 (pRpRkM-EBES-K(Biotin)), and LMS13 (rTkWiK-EBESK(biotin)) show cells covering the entire bead surface, indicating interaction with the unique
compounds on 3 different beads. Rosetta shaped binding was observed which is as a classical
configuration found when compounds bind with high affinity. After the initial screening, those
individual beads which showed binding ability were then selected and rescreened to confirm their
significance. The peptides found on the beads were then sequenced using the Edman degradation
process to determine their amino acid sequence, and then subsequently analyzed by MALDI-TOF
mass spectrometry to confirm their sequence. In order to test for the specificity of binding, cells
were treated with glycoside hydrolases to cleave off all surface glycans resulting in the depletion
of the binding activity of the selected peptides with the cells using the OBOC assay.
25
A
B
Figure 6. OBOC screening of LMS peptides. Bead screening of ZR-75-1 breast cancer
cells identified the peptides designated as LMS 3, 9, and 13. Arrows in Panel A depict
beads LMS 3 and 9 which are on the left and right, respectively. The arrow in Panel B
points to the bead with LMS 13. Cells form a rosette shaped pattern when bound. Images
were captured under a normal phase-contrast light microscope at 100x.
26
Further glycomic studies conducted by Dr. Labrilla’s laboratory confirmed peptide binding to
high-mannose glycans using mass spectrometry.
Three dimension cell culture
After determining that the LMS peptides could bind to the ZR-75-1 cells using a 2D
model of screening, we wanted to screen them in 3D culture systems. The Lam lab has
developed a 3D culture system that allows us to better assess cell growth, morphology, and
organization, to determine if the LMS peptides have any biological activity besides binding as
seen in the 2D model. Initial screenings of the MCF7 cell lines were conducted using 3D liquid
suspension cultures without the addition of peptides. The MCF7 cells were used because they had
been previously shown to grow as mammospheres in 3D culture on ultra-low attachment plates
by Stem Cell Technologies. A 3D mammosphere culture kit was purchased from Stem Cell
Technologies for the 3D liquid culture experiments, and was used as a reference to compare to
our matrix-assisted cultures of ZR-75-1 cells. Figure 7 shows the visual difference in cellular
morphology found in different subtypes of the MCF7 cell line which were quite varied. It should
be noted that cellular, morphological and organizational changes are not normally apparent in 2D
culture. These 3D cultures show similar spheroid formation as embryoid bodies found during
hESC liquid suspension cultures.
These results were then compared to the culturing of ZR-75-1 cells in specially
formulated 3D matrix-assisted cultures which does not have the same limitations as liquid culture.
The 3D liquid culture technique is limited by its inability to change medium which thereby does
not support long-term culture and drug studies. The use of our unique hydrogel allowed us to
keep the cells in a long term culture, as well as allowed us to directly conjugate the LMS peptides
27
A
B
C
Figure 7. Mammospheres in 3D liquid suspension. Various MCF7 cells in liquid 3D
suspension cultures were imaged at 200x magnification under a normal phase-contrast light
microscope. Colonies were imaged after one week in mammocult medium so as to
generate mammsopheres. Panel A, MCF7 C6 cells in a colony. Panel B, MCF7 HER2
positive cells in a colony. Panel C, shows MCF7 cells in a colony.
28
to the PVA backbone taking advantage of the cis-diol hydroxyl groups on the PVA. These
hydroxyl groups allow for both stable yet reversible crosslinking and biotin conjugation, which
allows us to bind biotinylated peptides to the biotinylated PVA using streptavidin as an
intermediate.
The initial cultures of our ZR-75-1 cells in our matrix-assisted 3D culture system were
used to determine if hydrogel could support long term growth of the cells. In addition, cells
were, assessed for natural growth, morphology, and cellular organization. ZR-75-1 cells were
cultured in an 8-well culture slide using 5% crosslinker and 4% PVA. The results showed that
this PVA based hydrogel efficiently provided solid culture support for mammalian cells for up to
one month. Following culture medium changes, the gel weakened and fully dissociated after two
weeks time. Cultures could likely have been sustained longer if additional crosslinker was added
during medium changes. Figure 8 shows that mammosphere formation was clearly visible by 48
hours during the culture process. Spheroids grew consistently throughout the 5 weeks of culture
time and morphological changes could be seen throughout the assay. Cellular differentiation and
clear organizational changes were be seen by the third week. When PI staining was conducted
during the fifth week to assess the amount of death, minimal amounts of cell death was seen
throughout the colonies. Sustained growth and proliferation of the cells was observed within the
hydrogel.
29
A
B
C
D
E
F
G
Figure 8. ZR-75-1 mammospheres in PVA
hydrogel. ZR-75-1 cells were cultured for 5
weeks in PVA hydrogel and imaged using a
normal phase-contrast light microscope
overtime. Panel A, day 1(200x). Panel B, 48
hours (100x). Panel C, 1 week (100x). Panel
D, 2 weeks (100x). Panel E, 3 weeks
(100x). Panel F, 4 weeks (40x). Panel G, PI
staining at 5 weeks (100x).
30
After establishing the long term growth potential of the matrix-assisted 3D culture
system, the synthetic LMS peptides identified by the bead binding techniques were incorporated
into the hydrogel to test their effect on the cells. Since the hydrogel is comprised of biotinylated
PVA molecules, streptavidin was used to bind biotinylated peptides to the PVA so as to
incorporate them into the hydrogel. ZR-75-1 cells were then cultured in the hydrogels with
peptides incorporated to assess any growth or morphological effects. Figures 9 through 12 show
the affects of LMS3, LMS9, and LMS13 on ZR-75-1 cells in the hydrogel. Both LMS 3 and 9
peptides seem to have growth inhibitory and apoptotic affects on ZR-75-1 cells. The cells did
have minimal amounts of growth as compared to the negative control, and large mammosphere
formation seemed to be absent. PI staining was done on each well to determine the amount of cell
death. As seen in Figure 11, PI staining confirms the apoptotic effect of LMS 3 and 9 on the cells.
LMS 13 showed similar growth and apoptosis levels as the negative control which indicated that
it had no effect. It should be noted that each of the treatments did have cell growth, but only the
negative control and LMS 13 wells had continuous cell growth throughout the 5 weeks of culture.
31
A
B
C
D
Figure 9. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides at Time zero.
Initial time point of ZR-75-1 cells cultured with PVA hydrogel incorporated with LMS 3,
9, and 13 peptides. All images were captured using a normal phase-contrast light
microscope at 40x. LMS 3, 9, 13, and negative control are shown as A, B, C, and D,
respectively.
32
A
B
C
D
Figure 10. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides after one week.
Week one of ZR-75-1 cells cultured with PVA hydrogel incorporated with LMS 3,9, and
13 peptides. All images were captured using a normal phase-contrast light microscope at
40x. LMS 3, 9, 13, and negative control are shown as A, B, C, and D, respectively cluster.
33
A
B
C
D
Figure 11. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides after three
weeks. Week 3 of ZR-75-1 cells cultured with PVA hydrogel incorporated with LMS 3, 9,
and 13 peptides. All images were captured using a normal phase-contrast light microscope
at 40x. LMS 3, 9, 13, and negative control are shown from A, B, C, and D, respectively.
34
A
B
C
D
Figure 12. ZR-75-1 mammospheres in PVA hydrogel with LMS peptides after 4 weeks. PI
staining of ZR-75-1 cells cultured for 4 weeks with PVA hydrogel incorporated with LMS
3, 9, and 13 peptides. LMS 3, 9, 13, and negative control are shown from A, B, C, and D,
respectively. PI photos taken with a fluorescent microscope at 488nm excitation and
570nm emission.
35
In vivo xenograft and nanoparticle biodistribution studies
The next step of my project was to assess the biodistribution of the nanocarriers without
LMS peptides in a mouse model to serve as a baseline. This was done to determine if there was
any targeted uptake of the chemotherapeutic when the LMS peptides are used in combination
with the nanocarriers. A xenograft mouse model was made for these studies using ZR-75-1
human breast cancer cells. The model was created by injecting 5x106 ZR-75-1 cells into female
nude mice. Injections were done subcutaneously in the lower right flank using ZR-75-1 culture
medium without fetal bovine serum (FBS). Successful tumor uptake was visible after one week
time period. Figure 13 shows an image of one female nude mouse with visible tumor growth in
the lower right flank after injection.
36
Figure 13. Nude female mouse with ZR-75-1 xenograft. Cells were injected
subcutaneously into the lower right flank and photos taken after one week.
mass.
, tumor
37
After the tumor grew to a sufficient size, biodistribution studies were conducted using
drug-loaded (doxyrubicin) superparamagnetic iron oxide (SPIO) nanoparticles. The drug-loaded
particles were then injected intravenously through the tail vein, intra-tumoral, and intra-peritoneal
regions and then analyzed for tumor targeting by in vivo fluorescent imaging. Figure 13A shows
images of drug-loaded SPIO targeting within the mice. The bright gray highlighted areas show
the location of the drug-loaded nanoparticle. As seen in Figure 14A, high levels of targeting is
seen at the tumor site, with nonspecific targeting observed in the abdominal region. Tumor
targeting was observed in all mice examined. Out of the intravenous tail vein injections, the intratumoral injections, and the inter-peritoneal injections, the intravenous tail vein injections had the
best targeting toward the tumor. This injection method, had the most drug-loaded SPIO in the
tumor region and less nonspecific uptake when imaged. The mice were then sacrificed and
analyzed by necropsy as well as ex vivo imaging to assess the biodistribution of the SPIO
particles. Figure 14B shows the biodistrubution of the drug-loaded nanoparticles using
fluorescent imaging. Most of the nanoparticles ended up in the tumor site, but some nonspecific
uptake was seen in the liver and kidneys. The intestines did show some fluorescence, but this was
thought to be due to the mice not undergoing fasting before imaging. These results show that
drug-loaded nanoparticles have high levels of tumor targeting capability due to the enhanced
permeability and retention effect, even without additional targeting ligands or magnetic resonance
localization.
38
A
B
Figure 14. Nude female mice with ZR-75-1 xenograft, and SPIO biodistribution. Nude
female mice with ZR-75-1 xenograft tumors injected with Superparamagnetic Iron Oxide
particles and their in vivo and ex vivo biodistribution. Panel A, in vivo mice image with
SPIO biodistribution. Panel B, ex vivo mice organs image with SPIO biodistribution.
Kodak IS2000MM Image station with a 625/20 band pass excitation filter, 700WA/35
band pass emission filter, and 150 W quartz halogen lamp light at maximum.
39
DISCUSSION
Human disease and cancer in particular is one branch of biology that continues to grow
with information and in return, complexity. It is clear that most multifaceted and multi-factorial
diseases such as cancer will need to be treated with new approaches that take new insights into
consideration. When focusing particularly on cancer, one has to consider many aspects including:
cancer progression, the tumor microenvironment, the heterogeneity of the tumor, and its
interaction with the rest of the human body. When doing so it becomes quite clear that the only
way to truly, efficiently, and successfully treat a cancer patient is with combination therapy.
Combination therapy is a therapeutic approach that targets the many features of a
malignant tumor and treats the patient with therapies that are both tumor specific and general. The
goals of this project were to develop new research methodologies and therapeutics to overcome
the limitations of current therapeutics and improve the therapeutic index and efficacy/toxicity
balance when treating breast cancer patients. With the use of the OBOC method, nanotechnology,
and novel 3D culture screening models we have developed a new approach to discovering
therapeutics, screening these therapeutics, and directing these therapeutics towards the tumor
during treatment.
Glycans have been shown to alter the pathology of cancer progression, contributing to
many of the required factors of malignancy (3). Therefore, studies were conducted by Dr.
Lebrilla’s lab to analyze the glycome profile of several breast cancer cell lines. These studies
found high-mannose glycan expression to be elevated in breast cancer cells as compared to
normal breast cells. Specifically, nine branched mannose residues were found which are the type
that are added early during N-linked glycosylation. The biosynthesis pathway of N-linked
40
glycosylation has an intermediate step of high-mannose glycosylation before alteration into
complex/hybrid glycans. N-linked glycosylation starts early on in the endoplasmic reticulum, and
high-mannose glycans are particularly important during protein folding and protection against
degradation during intracellular transport. This aberration in high-mannose glycan expression
suggests a problem early on during the biosynthesis pathway. Possible due to genomic mutations
and increasing instability this normal process is altered and high-mannose glycans end up
becoming the majority of surface glycans. Another possibility is that the expression of these highmannose glycans could give these cells a growth advantage, allowing selection of these cells to
proliferate and become increasingly genomically instable. This mutation can greatly affect the
proteins being produced by causing improper folding, increased degradation, and altered
functionality. These studies revealed that glycans can serve as a new biomarker and target for
malignant breast cancer. This glycan profile on the surface of the cell might also be playing a role
in the molecular pathology of the cancer progression, including contributing to the “stemness” of
the cancer cells.
When comparing MCF7 HER2 and C6 cells against H1 cells that have a 74% expression
level, it’s interesting to note the similarities in expression and cell capability. Both cell lines are
highly proliferative, can self-renew, and the cancer cells might have multi-potential allowing
differentiation of cancer cells into other cells types. Also to note, both MCF7 cell lines were
analyzed by flow cytometry and found to be positive for the CD44 surface marker and negative
for the CD24 marker which is characteristic of breast cancer stem cells, further arguing in favor
of the cancer stem cell hypothesis (12). Identical expression of these surface molecules on stem
cell and aggressive cancer cells provides more evidence in favor of the cancer stem cell
hypothesis. Interestingly, the BT549 cells did not show an increase in the amount of high-
41
mannose glycan expression, but rather a decrease. However, this data does not disprove the fact
that high-mannose glycans are good targets for therapeutics, but instead reaffirms the notion that
we still need to to discover these differences in tumor subtypes and treat patients accordingly.
Overexpression of high-mannose glycans in malignant breast cancer offers a very unique
and specific target for targeted therapeutics. The particular targeted therapeutics for breast cancer
treatment can be discovered by the use of the OBOC screening method. Our project has shown
the ability of the OBOC method to discover new novel therapeutic agents. It has helped us
discovery three synthetic peptides (LMS 3, 9, and 13) that can directly target high-mannose
glycans. Malignant breast cancer cells that have high levels of surface high-mannose glycans
were found to bind directly to these peptides. These peptides should be able to take advantage of
the high levels of high-mannose glycans on cancer cells so that cytotoxic drugs can be delivered
when the peptide ligands bind to the cells.
These peptides have also been shown to have growth inhibitory effects on breast cancer
cells when assayed using our biotinylated hydrogel which is an easy and efficient system to
screen potential therapeutics. Each of the LMS peptides was screened in a 3D culture model using
the hydrogel for an extended amount of time, making this culture system not only good for long
term culture assays, but a useful tool in drug discovery and testing. The 3D culture model
provides an opportunity to mimic the in vivo response of breast cancer cells to the presence of
these peptides due to the interactive three dimensional space (6). The results demonstrated that
both LMS 3 and 9 peptides inhibit growth and mammosphere formation in 3D culture. These
results show that some LMS peptides will help not only in targeting, but can also participate in
the destruction of the resistant/stem-like cancer cells. Future studies will need to be conducted to
42
determine the exact pathways being altered, but current data suggests that these peptides are very
good candidates for our combination therapy system. In combination with nanoparticles, we can
selectively deliver several therapeutics directly to the tumor site, allowing for deep penetration
and increased concentration over time. Results from these analyses indicate the potential
therapeutic effects of the LMS peptides, and the usefulness of our unique 3D culture model.
3D cultures provide a more physiologically similar method to analyzing cell growth,
interaction, gene expression, and response to drugs (13). More specifically matrix-assisted 3D
cultures that suspend the cells within a solid to semi-solid medium allows for increased
effectiveness in long term culturing and drug screening. Our 3D culture method utilizes
biotinylated PVA as a backbone and PEG conjugated boronic acid as crosslinkers, forming a
hydrogel. Hydrogels are especially unique because they are mainly composed of water and allow
culture medium diffusion and absorbance. The boronic acid crosslinkers bind to cis-diol hydroxyl
groups on the PVA backbone. This conjugation is flexible and can be reversed with an addition of
a competing compound such as fructose, or decreasing the pH. Biotin conjugation of PVA allows
for rapid and strong binding of any other biotinylated compounds or molecules to it using
streptavidin to link them together. Novel therapeutics such as glycan binding/inhibiting ligands
can be screened in this culture method to better predict the in vivo outcome and effectiveness. The
results attained from the initial 3D culture validate the significance of using 3D modeling for
many fields of biology, including drug discovery.
As stated previously, additional studies will need to be conducted to determine the
molecular mechanism of the growth inhibitory effects found in the LMS peptides. When
answered, studies can then performed to incorporate these peptides into nanoparticle drug
43
delivery systems and test to see if they will have a high therapeutic index. SPIO nanoparticles
provide a promising avenue for drug delivery (14). Not only unique in their ability to be localized
by magnetic resonance, and contribute to therapy by site-specific heat induction, but the presence
of dextran sulfate on the particle allows for conjugation of our LMS peptides. Combination of the
LMS peptides with the nanoparticle drug delivery system could greatly increase the intra-tumoral
concentration of these therapeutics, and reduce any side effects caused by nonspecific uptake. We
conducted initial tests of the biodistribution with drug-loaded SPIO particles. These tests assessed
the tumor targeting capability of the drug-loaded nanoparticles alone before the addition of the
targeting peptides. These initial tests show a superb capability of the drug-loaded SPIO
nanoparticles to target the tumor alone. These results provide a sound argument for the use of
SPIO nanoparticles in cancer therapy, and for inclusion as a candidate for combination therapy.
Future studies will assess the toxicity and therapeutic effect of the drug-loaded SPIO particles
with and without the LMS peptides. Also, the SPIO nanoparticles will facilitate studies to
examine the biodistribution and toxicity since they can be localized by magnetic resonance, and
should also contribute to the therapeutic index since they can be heated up to kill cells. The use of
all of these combined therapeutics and methods should provide a unique effective way of treating
breast cancer patients, both efficiently and safely.
In summary, it is clear that with the use of novel in vitro and in vivo therapeutic screening
we can successfully discover new therapeutics that will help save lives and improve the quality of
life during treatment. Screening OBOC libraries in vitro for tumor targeting has helped us
discover novel glycan binding peptides, which also show growth inhibitory effects in 3D tumor
modeling cultures. The LMS peptides are unique in their targeting and functional abilities. New
glycomic studies have shown the importance of high-mannose glycans in cancer progression.5,6
44
These glycans are highly over expressed and contribute to all six hallmarks of cancer (3). Using
the LMS peptides to directly bind to these novel targets is an exceptional way to use the cancers
own weapons against it. Screening of these peptides in a 2D model and 3D model has given us
the ability to examine additional functional capabilities. Not only will targeting help, but
functionally these peptides show growth inhibitory effects, which can help the therapeutic
process. The biodistribution analysis of our drug-loaded nanoparticle system has shown success
in targeting the tumor mass. Both in vivo and ex vivo imaging studies confirm the high levels of
tumor uptake. The next step of the project will be to analyze the biodistribution and therapeutic
index of the entire drug delivery, and combination therapeutic system. Addition of the LMS
peptides to the nanoparticles should increase the targeting capability and decrease any nonspecific targeting. Success in the initial phase of developing this new novel combination therapy
is a big step in cancer therapeutics and will help pave the way for future therapies.
45
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47