1. From Breast to Bone: A Study of Breast
Cancer Metastasis Using Tissue
Engineering
Chris Tippins, Gus Huffines, Leigh Karas and Jenna Alsaleh
December 4th
, 2015
Fall 2015

2. Abstract:
Breast Cancer is still one of the most diagnosed cancers in women and while treatments can
often help in situ tumors, the metastasizing effects are still incurable and deadly. The mechanisms via
which breast cancer metastasizes into other tissues is not completely understood. We describe an
experimental method to study these mechanisms by designing six three-dimensional (3D) scaffold
models to simulate adipose tissue and bone tissue. These six scaffolds will be split into three adipose
tissue models and three bone tissue models and will be developed using a scaffold material that allows
for a variation of stiffness. The scaffolds will consist of a chitosan-collagen hybrid crosslinked with
genipin. The scaffolds will be developed Metastatic cancer cells from the MDA-MB-435 cell line will be
seeded onto the fully developed and vascularized scaffolds and studied for changes in genetic
expression, histology, and chemical indicators of metastatic cancer. More specifically, an examination
of the presence of urokinase-type plasminogen activator system (uPa) and serpin inhibitor plasminogen
activator inhibitor 1 (PAL1) along with polymerase chain reaction (PCR) testing for genes associated
with cancer metastasis will be tested to attempt to elucidate the metastatic cascade. By using a tissue
engineering approach, we will avoid the pitfalls of using either patient tissues or non-human models
while developing a robust manufacturing methodology for bone and adipose tissue for use in other
tissue engineering pursuits. By improving the basic understanding of how breast cancers metastasize
to bone tissues, we hope to improve overall patient outcomes by facilitating pathways to new
opportunities for genetic testing, prevention, and after-the-fact treatment strategies.

3. Specific Aims:
Breast cancer metastasis to bone is a major problem for breast cancer patient outcomes.
Currently, the mechanisms by which cancer is able to bypass bone tissue defenses, invade the tissue,
and proliferate once there are unknown. Many theories on the chemical and signal cascade that allows
for metastasis have surfaced, but the only way to create effective therapeutics is to understand this
process. Developing this understanding requires modeling the microenvironments involved. Our
objective is to create an in vitro model of adipose tissue that mimics the environment of breast tissue
enough to allow cancer cells to attach and proliferate. Once successful, we can then change the
stiffness and environment of the scaffold to mimic native bone and study any tumor changes. These
models could then be used for various studies on metastasis as well as for tissue engineering
manufacturing studies. We hypothesize that there will be a significant difference in breast cancer
proliferation and adherence as the stiffness of the hydrogels change and that there will be a difference
in the physiological state of breast cancer based on the tissue environment the cancer is located within;
different signaling molecules will require different survival strategies
Aim 1: Scaffolds
We aim to make hydrogels of varying stiffness for each of the tissue cell lines seeded to study
the effect that varying microenvironment stiffness has on metastatic breast cancer. We will use a
chitosan-collagen hybrid crosslinked with genipin. We hypothesize that by changing the cross-linking
time and/or concentration we can pinpoint the gel stiffness that mimics the elastic modulus of bone,
adipose and breast cancer tissue.
Aim 2: Cell Lines
Two types of cell lines are of interest for this project: A human mesenchymal stem cell line for
differentiation into both adipose and bone tissue and a highly metastatic breast cancer cell line for study
of the metastatic cascade. We hypothesize that each of these cell lines will thrive and develop within
our scaffolds and that examination will show differentiation of the mesenchymal stem cells into their
respective tissues.
Aim 3: Bioreactors
We aim to create a bioreactor with different microenvironments suitable for both adipose and
bone tissue scaffolds that mimic the in vivo environment as accurately as possible. By using growth
factors such as insulin-like growth factor-β (IGF-β) and bone morphogenic protein (BMP), as well as
fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), we can create the right
environment for cell growth, differentiation and proliferation. We hypothesize that with the right
mechanical stability and correct use of growth factors, we can create accurate representations of the in
vivo microenvironment.

4. Background Information:
What Is Breast Cancer Metastasis?
In the United States, from 1999 to 2011, female breast cancer held the number 2 position in
cancer incident rates per year [1]. According to the CDC, in 2012, female breast cancer moved to the
number 1 position with an age adjusted incident rate of 122.2 cases per 100,000 women [1]. With an
estimated 40,290 women expected to die in 2015 from breast cancer and U.S. care spending projected
to break $25 billion per year by 2020, research into the disease’s causes and treatment are critical to
the population [2, 3].
Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal
cells [4]. Cancer of the female breast tissue is one of the most common types of cancer in the United
States. There are several different types of breast cancer but the most common are ductal carcinoma
and lobular carcinoma with ductal carcinoma being more prevalent [5]. All cancers start in a specific
tissue and many times when detected early, can be successfully treated. In fact, stage 0 breast cancer
has a 98% 5-year survival rate[4]. In ductal carcinoma, the abnormal cells begin growing on the lining
of the milk ducts and at this stage, the cancer is usually described as what many know as a “lump” or
stage 0 breast cancer [5]. In lobular carcinoma the concept is similar and in both cases, if the lump is
detected early, the chances of successful treatment are higher. If left untreated or detected, these in
situ (meaning in the original place) carcinomas can spread to other tissues [5]. This spreading to other
tissues such as bone or the lungs is referred to as metastasis and is usually in the final stages of breast
cancer (stage IV or TNM) [5]. Because the cancer is so invasive at this stage, metastatic breast cancer
is the most deadly stage of breast cancer with 5-year survival rates at a staggering 24% [4]. Due to the
high number of variables such as age, cancer recurrence and life expectancy, it can only be estimated
how often breast cancer will become metastatic. According to one study, 20-30% of all breast cancer
cases will become metastatic [6]. And according to the American Cancer Society, of those metastatic
cases, 2 out of 3 will metastasize in the bone [7].
Bone metastasis occurs when cells from a cancerous tumor break off, are circulated through the
body via lymph fluid or blood and settle on a new tissue, usually bone [3]. Once on these new tissues,
they can grow into new tumors and have negative effects on the new host tissue. In bone metastatic
breast cancer, the cancer cells often release substances that activate osteoclasts to unnaturally high
levels causing bone atrophy or osteoblasts leading to unnecessary bone growth both resulting in
extreme pain and brittle bones [3]. While treatment for bone metastatic tumors is available, it usually
only reduces pain and prolongs the life of the patient until damage to the skeletal system is to the
extent that the production of blood cells is inhibited or the protective mechanisms of bones are no
longer functional resulting in skeletal morbidity.
Since bone metastatic breast cancer is the most deadly and most commonly metastasized
breast cancer, new research into treatments to prolong life and improve the quality of life are in high

5. demand. Some of the most recent research has been directed towards the mechanisms by which the
tumor cells escape into the lymph or blood fluids, attach and survive, but these processes are poorly
understood.
Current Treatments for Breast Cancer Metastasis to Bone
As the metastasis to bone tissue is the most common form of breast cancer metastasis [8],
effective treatments are an important factor in reducing morbidity and mortality in breast cancer
patients. These treatments aim to reduce skeletal injuries such as fractures and pain to improve patient
quality of life. The most important treatment for bone metastases is intravenous bisphosphonate
therapy– a chemical inhibitor of osteoclasts, which are cells that absorb bone tissue during cycles of
injury healing and regrowth [8]. This inhibition includes inducing apoptosis in osteoclasts as well as
preventing or slowing differentiation and growth [8]. Bisphosphonate may also have a hand in
destroying cancerous tumor cells themselves [8]. However, bisphosphonate treatment has serious
drawbacks when used in patients: renal toxicity, gastrointestinal upset, and others [8]. Bisphosphonate
therapy is often used in conjunction with other cancer treatments such as chemotherapy, endocrine
therapy, radiotherapy, and others. There is generally a better patient outcome when bisphosphonate is
used with these other therapies when compared to use of bisphosphonate alone or non-use of
bisphosphonate [9]. There are studies ongoing into the genetic predisposition of certain cancer cells to
metastasize to predict which cancer patients will experience metastasis [10].
Breast Cancer Metastasis Prevention
Currently, the cellular mechanisms of tumor metastasis is poorly understood. However, there is
some evidence that calcium and vitamin D may play a role in breast cancer metastasis, particularly in
reducing inflammation [11] . Inflammation is a central part of the immune response to infection and
injury, and there is overwhelming evidence that it plays a role in the development of cancer [12]. In
fact, cancerous cells have been shown to have genetic changes that promote inflammatory chemical
pathways and tumors have been observed attracting fibroblasts and macrophages, which are common
during the inflammatory response [12]. Thus, many cancers that are said to be caused by infectious
agents, such as cervical cancers caused by the human herpes virus-8 (HHV-8), are caused in part by
the inflammatory response recruited by the immune system’s response to infection [12]. Thus, it is
obvious that reducing inflammation is key in preventing the development or worsening of
cancers. There is evidence that taking non-steroidal anti-inflammatory drugs (NSAIDs) such as
ibuprofen, aspirin, and others reduces relapse and mortality in breast cancers [11]. Other chemical
pathways to reduce inflammation have also been explored; for example, vitamin D has been shown to
reduce cancer cell proliferation. Its active chemical component, 1,25-dihydroxy vitamin D, has been
shown to chemically reduce inflammation as a natural immune control in a healthy human [11]. Thus,
vitamin D can also be considered immunosuppressive [11]. Conversely, vitamin D deficiency has been

6. shown to cause the recruitment of breast tumor cells to the bone, leading to breast bone cancer lesion
development [11].
The Mechanical Strength of Breast and Bone Tissue
When creating both adipose and bone tissue models there are numerous assumptions and
many biochemical, mechanical and other factors that play a role in how accurate an in vitro model will
be. The differences in these factors for each tissue type affect strongly the interactions and morphology
of breast cancer as it metastasizes. Unfortunately, it is nearly impossible to determine from in vivo
studies which factor or factors is the cause of a particular change in cancer morphology. This is why in
vitro studies are a fantastic way to study individual differences between the tissues in a controlled
manner.
For our study, we will measure the differences in the mechanical strength of breast and bone
tissue and analyze how that plays into breast cancer morphology and progression. A study done by the
Mayo Clinic on six breast cancer patients concluded that on average, breast adipose tissue had an
elastic modulus of about 3 kPa while the tumor’s modulus was approximately 12 kPa [13]. Bone tissue,
obviously, is much stiffer, with one study measuring a Young’s modulus of 20 GPa, which is higher than
both breast and tumor tissue stiffness [14]. Since tumor tissue is often stiffer than breast tissue, we
hypothesize that the similarity in stiffness between tumor and bone may aid in breast cancer’s
metastasis to bone. Thus, we will take stiffness as our factor to study when designing our tissue
models.
Current Models for Breast Cancer Metastasis to Bone
To study the metastasis of breast cancer to bone, in vitro two-dimensional (2D) models are the
simplest means to screen for genes and proteins which are major players in the mechanism. Although
this method lacks the physiological complexity of in vitro 3D models and in vivo testing, it is a great
starting point for any preliminary study. The relatively low cost and more well-established 2D systems
normally precede the use of more expensive and complicated 3D models. High-throughput screening
analysis using 2D systems help narrow down the amount of tests that would have to be carried out
using 3D. Indeed, three-dimensional models that can be produced both reliably and cheaply will
eliminate the need for using 2D models for preliminary studies. Furthermore, 3D models may provide
insight into more intricate details of certain mechanisms that were overlooked in the 2D screening
analysis, potentially leading to more discoveries. As a result, 2D models have had their role in this area
of research, but today many researchers in the field focus on designing 3D models to produce
environments that are as close to in vivo conditions as possible.
In recent years, scientists have shifted focus to using 3D models as a way of modeling the
metastatic cascade. This allows a better mimicry of the in vivo environment that surrounds bone and
cancer cells. To study and understand the metastatic cascade, scientists must be able to identify and

7. measure the ability of a tumor to adhere, migrate, invade, proliferate and survive [15]. 2D models lack
the complexity of the in vivo environment which makes it difficult to determine whether administered
treatments that work on the 2D model will work in the actual environment.
One type of model that has been used recently is that of Hydrogels. Hydrogels contain a large
amount of water and contain many naturally occurring molecules like collagen and fibrin. Dr. Zhu and
colleagues from The George Washington University used chitosan hydrogels to model breast cancer
metastasis to bone [16]. Chitosan is a polysaccharide commonly found in crustaceans but it contains
sugars that are good for creating bone scaffolds. The team used 3D porous chitosan bone scaffolds
containing hydroxyapatite (a component of the bone extracellular matrix). They also used human bone
marrow mesenchymal stem cells to deposit bio-active factors within the scaffold to create a more
biomimetic environment [16]. Three different kinds of breast cancer cell lines, each with different levels
of metastatic activity, were seeded on different scaffolds to evaluate the bone scaffold. The researchers
discovered that breast cancer cell adhesion and proliferation increased with decreasing hydroxyapatite.
They also discovered that their selected model is able to simulate breast cancer metastasis using
various cancer cell lines. Their scaffold could provide a reproducible and controllable platform for
identifying potential therapeutic gene targets as well [16]. While a large improvement to 2D models,
there is still a question, however, on whether the model is accurate enough to compare to in vivo
environments. This requires more intensive study and improvement upon the scaffold.
In 2014, a collaborative research group worked on a project using a microfluidic system to study
the extravasation of breast cancer into the bone cell – matrix environment [17]. Their microfluidic model
consisted of three media channels and four independent gel channels. The gel channels were
embedded with osteo-differentiated human bone marrow mesenchymal stem cells. They also created
channels without human bone marrow mesenchymal stem cells for control purposes. The channels
were injected with human breast cancer cells as well as lined with endothelial cells [17]. The
researchers noted that due to the ability of extravasation of about 37% in the collagen-gel only matrix,
that matrix density is not a major player in metastasis of breast cancer. The cancer cells also travelled
greater distances in the extracellular matrix compared to collagen gel only models suggesting that ECM
density plays a role in extravasation [17]. The researchers did mention, however, that the model was
not a perfect representation of what happens in vivo. For instance, bone remodeling and changing of
the extracellular matrix is a typical action in vivo. If the osteo-cells were cultured longer in the
microfluidic model extracellular matrix changes could occur and influence cancer cell behavior [17].
Chemokines and growth factors are essential parts of cell-cell and cell-matrix interactions. A better
study of the ability for secretion of these chemicals by osteoblasts could improve knowledge in the
metastatic cascade.
There have been a few problems with using 3D matrices while studying cancer metastasis.
Some of the problems with current 3D models to study the metastatic cascade have been the use of

8. natural matrices such as Matrigel which can contain growth factors not found in natural bone and can
influence cell migration [15]. In the last few years, however, a development of synthetic polyethylene
glycol-, hyaluronan or alginate-based hydrogels have been used to study integrin binding, chemokine
and growth factors [15]. These gels have an adjustable stiffness that scientist can modify to study the
effect of rigidity on metastasis. Another issue includes the cost and availability of 3D matrices when
compared to that of 2D matrices. Most 3D scaffolds also require long culture periods to study the true
pathway of cancer as well as use materials close to real tissue. This time-dependency can sometimes
harm a researcher’s ability to use or create a novel model.
Significant leaps and bounds into the ability to model breast cancer metastasis to bone have
been made within the last decade. While there are advantages to using 2D analyses, it seems that the
field has an overwhelming shift to three dimensional models, with good reason. Three dimensional
models better mimic the environment of breast cancer metastases than two dimensional models. The
trick is to create a model that is able to portray the in vivo environment as accurately as possible while
still being able to control said environment.
It is hopeful that by studying the extracellular matrix and signaling pathways of cells in the bone
and breast cancer environment as well as through detailed use of biomaterials and tissue engineering
principles that a better model of the metastatic cascade will be created for future use. This proposal
has the potential to revolutionize the understanding of breast cancer metastasis to bone by elucidating
biochemical processes that occur when tumor cells come in contact with bone or adipose tissue. It is
only via tissue engineering that we can ethically and precisely model the metastatic cascade.
Understanding how cancer metastasis happens is a powerful tool; the more we understand, the better
we can design our therapies and the closer we are to curing Breast Cancer!
Research Design:
The overall goal for our experiment is to test mechanical strength and microenvironment on
tumorigenesis of metastatic breast cancer. Hydrogel scaffolds made out of a collagen-chitosan hybrid
cross-linked with Genipin will be created. By changing cross-linking techniques, we aim to create
scaffolds that mimic the elastic modulus of breast tissue, bone tissue and breast tumor tissue. Once
this is done we want to seed mesenchymal stem cells on these three scaffolds that mimic an adipose
environment and use signaling factors to induce adipose differentiation. We then want to use three
more of these scaffolds, seeded with the mesenchymal stem cells, and add growth factors that induce
bone tissue differentiation. After analysis, we finally want to add a metastatic breast cancer cell line to
each of these six scaffolds and analyze how the mechanical stiffness and environment coincide to
create differences in the morphology and proliferation of metastatic breast cancer. The overall
schematic for the design of this experiment can be seen by Figure 1 at the end of this paper.

9. Specific Aim 1: Construction of Hydrogels with Varying Stiffnesses:
Our team first aims at creating hydrogels made out of materials that allow for a change in
stiffness. This allows for control so that we can create both soft adipose tissue and hard bone tissue
using the same scaffolds. Therefore, differences between the scaffolds will be based solely on change
in tissue microenvironment and not on type of hydrogel used.
Current research shows that the use of chitosan-collagen hybrids as a hydrogel for bone and
adipose tissue engineering creates excellent porous structures with an ability to change the mechanical
strength in slight, controlled increments [18] and [19]. Freeze-drying is an effective and comparatively
easy technique in creating porous scaffolds for cell adhesion and proliferation. To change the
mechanical properties of the scaffolds, the cross-linkage between polymers can be varied. Adding more
cross-linking has shown to increase the compressive strength of the scaffold, which is measured as the
modulus of elasticity. We will design chitosan-collagen based scaffolds and cross-link the polymers
using Genipin, a natural cross-linker. Genipin has decreased cytotoxicity compared to glutaraldehyde, a
commonly used cross-linking agent, making it more suitable for studying tissues [20].
Our scaffolding techniques will be adapted from multiple research groups, most notably Wang et
al [20] and Annabi et al [18]. First, chitosan of a high molecular weight and collagen type I will be
dissolved in an acetic acid solution. These scaffolds will then be frozen at -20 degrees Celsius for one
day and freeze-dried to form porous scaffolds. They will then be immersed in Genipin solutions at
intervals of 10 minutes, 30 minutes and 1 hour for preliminary study of the efficacy of the Genipin. If
unsatisfactory results are achieved, then different concentrations by weight of the Genipin solution will
be used to determine the optimal method to achieve the goal compressive strength. The aim is to
create mechanical strength in each scaffold that is comparable to bone tissue, fat tissue and tumor
tissue. Stiffness can be changed by changing both time allowed for cross-linkage and concentration of
cross-linking agents such as Genipin. Multiple experiments and tests will be conducted to determine
which of these techniques is optimal for controlling the mechanical strength of the scaffolds. Our aim is
to get the scaffolds to mimic the mechanical strength of approximately 3 kPa (adipose tissue), 20 GPa
(bone tissue) and 12 kPa (breast tumor tissue) to model what actually happens mechanically in vivo
[21]. All scaffolds will be rinsed with phosphate buffered saline prior to cell seeding experiments and will
undergo porosity, modulus of elasticity and histological testing before being used for cell seeding
Specific Aim 2: Culture and Seeding of Mesenchymal Stem Cells:
Our second aim is to successfully culture and seed mesenchymal stem cells and breast cancer
tumor cells onto our hydrogels. After the chitosan scaffolds have been manufactured, they will be
seeded with a variety of cells to simulate healthy adipose and bone tissue as well as breast cancer
growth and metastasis mechanisms. Three primary tissues types will be cultured: adipose tissue,
bone/bone marrow tissue, and breast cancer cells.

10. Dulbecco’s Modified Eagle Medium (DMEM) will be used to culture the three primary tissue
types. Using the same culture medium between the three tissue types will create a consistent baseline
to eliminate any confounding variables that would arise by using completely different media for each
cell type. Indeed, the DMEM will be supplemented with various factors which are vital in the
proliferation of each cell type. These modifications are described in each tissue section.
The MDA-MB-435 cell line has been selected for metastatic cancer cell seeding as it is the most
metastatic line available. MDA-MB-435 cells are estrogen and progesterone receptor-negative,
metastatic, ductal carcinoma cells [22]. To maintain the culture of these metastatic cells, fetal bovine
serum will be added to DMEM to 10% and gas exchange will be regular atmosphere maintained at 37
degrees centigrade per instructions of supplier (ATCC) [23].
A human derived bone-marrow mesenchymal stem cell line will also be used for both adipocyte
and osteocyte differentiation onto the scaffolds. The American Type Culture Collection sells a hbm-
MSC cell line, PCS-500-011, that can be used for this purpose [23]. From human mesenchymal stem
cells, adipocytes can take anywhere from 7-21 days to differentiate while the osteocytes will completely
differentiate after 14-21 days in differentiation media [24]. As a result, both cell lines will be allowed 21
days to differentiate to achieve maximum differentiation.
Upon successful passaging and proliferation of each of the three cell lines, each of the scaffolds
will be molded into small petri dishes for each study used and maintained in the DMEM. Standard
incubation parameters will be met to keep the cells at optimal adherence and proliferation conditions.
Specific Aim 3: Stem Cell Differentiation via Bioreactors and Signaling Factors:
We finally aim to differentiate our mesenchymal stem cells into adipose and bone cells by using
signaling factors and bioreactors to successfully mimic the in vivo environment.
Adipose tissue bioreactor conditions:
To differentiate adipose-derived mesenchymal stem cells (ASC), 10% FBS, 393 nM insulin and
100 nM dexamethasone will be used. For bone marrow-derived mesenchymal stem cells (bMSC), 10%
FBS, 175 nM dexamethasone and 50 μM indomethacin will be used. For the research being conducted,
bMSC will be used for culture, differentiation and seeding onto scaffolds for adipose tissue. The ASC
method of adipocyte generation is only included for reference and to encourage a study comparing the
two methods for creating adipose tissue models. The bioreactor that will be used to seed and maintain
the adipose tissue constructs will contain the defined media described above to encourage cell
differentiation, proliferation and seeding onto the scaffold. Furthermore, vascular endothelial growth
factor (VEGF) will be supplied at a physiological concentration to promote angiogenesis in the tissue
constructs. A well-vascularized system is essential in providing the ideal microenvironment with which
to study tumor proliferation and metastasis in the tissue, especially as the main method of metastatic
tumor cell movement is through blood and lymph fluid.

11. Bone tissue bioreactor conditions:
To generate the osteocytes needed to seed the scaffold to create a bone tissue specimen,
bMSC will be used and differentiated with bone morphogenetic protein 2 (BMP 2), transforming growth
factor beta (TGF-β) and parathyroid hormone (PTH) to generate osteoblast progenitor cells. Once
differentiated, these will be seeded onto the scaffold and then maintained with a medium containing
BMP 2, 4 & 6 along with PTH, vitamin D, basic fibroblast growth factor (FGF) and insulin-like growth
factor 1 (IGF 1) which will encourage maturation of the osteoblasts in the scaffold [24]. Exact quantities
of these hormones and growth factors are proprietary information of the company from which the
osteoblast differentiation medium is supplied, and therefore will not be listed here. VEGF will be
supplied at physiological conditions to encourage angiogenesis in the tissue construct.
Bioreactor design:
Overall Scheme:
Medium reservoir → oxygenator → scaffold/tissue construct chamber → medium reservoir
Conditions: 21% oxygen, 5% carbon dioxide, 37°C
The bioreactor will follow the scheme outlined above. The medium will be perfused over the
scaffold/tissue construct at a rate of 5 mL/min.
Once the adipose and bone tissue constructs have reached maturity, the bioreactor conditions
will stay the same for each respective tissue to maintain physiological conditions. The only change to
the system will be the introduction of cancer cell lines to the medium reservoirs of the tissue systems.
As the goal is to determine the effect of tumor on each of the two systems, the conditions must remain
the same to minimize variables that effect change in the microenvironment of the tissue constructs.
Testing and Analysis:
We first want to attain histological data by using scanning electron microscopy to see the
surface structure of the varying scaffolds and the control. This will allow for topographical and
observational, qualitative analysis of whether the scaffolds are heterogeneous, have a rough surface
structure and are porous.
Porosity will be measured by liquid displacement with the scaffolds being immersed in a known
volume of ethanol. The porosity is then calculated using the following equation:
Porosity (%) = (Wet Weight - Dry Weight)(Density of Ethanol) (Volume of Scaffold)x 100%
Where the wet weight is that of the scaffold immersed in ethanol and the dry weight is the
weight of the scaffold before being immersed in ethanol. The volume of the scaffold can be determined
by squaring the radius of the scaffold and multiplying by the height of the scaffold.
Mechanical testing will be achieved using a mechanical testing system with an 800LM
instrument similar to the one used by Wang et al [19]. A uniform small tare load of 0.1 N will be used for

12. each of the scaffolds to ensure a precise, controlled measurement. Although the mechanical testing in
this study was used only for bone tissue which has a higher elastic modulus than breast tissue, the
same tare load value of 0.1 N has been used in another study [25]. Force and deformation data will
then be collected to create a stress-strain diagram for each of the scaffolds. The slope of this diagram
will be used to calculate the elastic moduli.
Cell counts of bone, adipose and breast cancer cells will be used for proliferation studies using
100 microliter samples, trypan blue and a hemocytometer. This will be done over the span of 1 week.
Real-time PCR will be used to test expression of several genes for adipose, bone and breast
cancer cells. Total RNA from the cells will be isolated using an RNEasy mini kit, which can be used with
10 units of RNase inhibitor (GIBCO) and 40 ng of RNA. The sequencing primers will be taken from
known differentiation and proliferation genes specific to each of the cell-lines, while another primer
taken from a control gene expressed by all cell-lines will be used for comparison.
Post-Tumor Cell Exposure Testing
After the 3D tissue cultures have been successfully seeded and incubated on the scaffold, the
behavior of adipose and bone tissue in response to metastatic tumor cells will be analyzed. We hope
that these models will mimic adult tissues in their biochemical, morphological, and genetic response to
the tumor cells. As stated before, six models will be created, three consisting of bone tissue and three
of adipose tissue. Amongst both tissue types, the mechanical properties of the scaffold will be varied to
investigate how the tumor cell colonization is affected in the transition from breast adipose tissue to
hard bone tissue. These transitional scaffold properties will shed light on the stages of breast cancer
metastasis to bone and will allow analysis of the genetic, signaling, and morphological changes caused
during these transitional stages. Each model will be seeded with equal amounts of the MDA-MB-435
cell line incubated previously and all environmental conditions imposed by the bioreactors will remain
the same as during the initial construction of the 3D models. If reliable vascularization is achieved in
the 3D models, then the cells should be seeded through the blood vessels as this is how most
metastasis begins in vivo [26].
Once seeded with tumor cells, the models will be tested for the presence of metastasis cascade
molecules, changes in gene expression, and morphological changes in the tissue. Enzyme complexes
that degrade the extracellular matrix (ECM), specifically the urokinase-type plasminogen activator
system (uPa), are often found in early stage metastases [26]. One of the vital components of the uPa
system is the serpin inhibitor plasminogen activator inhibitor 1 (PAI1), which has been shown to be a
reliable marker in predicting metastasis in lymph-node-tumor negative patients along with uPa
activity[26]. Thus, the activity of these two markers will be determined via ELISA at regular intervals
throughout the investigation.

13. Changes in gene expression are also currently used both in modeling of breast cancer
metastasis and prediction of patient prognosis. Over 70 genes have been identified as factors in the
promotion of metastasis [26] although it should be noted that many of these genes are known to be
present in the tumor cells early in the disease progression, and so the tumor cell line used should be
tested via PCR for a select number of these genes before being seeded and the 3D model should be
expected to show an amount of gene expression directly related to the number of tumor cells that have
colonized the model. An interesting possibility is that tumor stem cells exist that drive metastasis,
explaining why some cancer cells will metastasize and others will not. The before-and-after
comparison of these genes will address this possibility. As is common practice, DNA microassays will
be performed periodically to determine gene expression[26].
Finally, morphological changes will be studied by use of scanning electron microscope (SEM)
imaging and immunohistochemical staining. Slices of healthy pre-exposure tissue will be compared to
post tumor cell seeding tissue at regular intervals throughout the project after SEM images are taken.
These slices will be stained to identify Mammoglobin A and B, which is a breast cancer marker
expressed when no metastasis has taken place [27]. A stain for GCDFP-15 should also be applied as
it shows a specificity for breast cancer between 98 and 99% [27]. Positive myoepithelial staining,
where noncancerous tissue is stained and tumor tissue is left untouched, should also be carried out to
show the volume of cancerous cell colonization present [27].

14. Figure 1: Experimental Design Schematic
Aceticacid
CollagenI
Chitosan
Freeze -20ᵒC
Lyophilized24hrs
CollagenI/Chitosan
Polymer
3 kPa
20 GPa 12 kPa
SEM,
Porosity,
Mechanical
Strength Testing
Cell Seeding
Signaling
molecules
PCS-500-011
PCS-500-011
3 kPa 12kPa 20GPa
Adipose Bioreactor
Bone Bioreactor
Adherence and
Proliferation
Analysis, PCR
BreastCancer
Cell Seeding
PCS-500-011- Adipose Bioreactor
PCS-500-011- Bone Bioreactor
Adherence and
Proliferation
Analysis, PCR
21 Days

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