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Report for FRD Grant on “Mechanisms of Glioma Cell Migration”
PI: Chandra Kothapalli, Ph.D.
Department of Chemical and Biomedical Engineering
INTRODUCTION:
Uncontrollable cellular proliferation and the formation of localized or remote tumor sites
have long since characterized the cancer phenotype. Progressive acquisition of organs and
various vital systems often follows a seemingly benign growth of tumorous cell types. As these
bodies continue to metastasize and proliferate, processes imperative for the continuation of life
become unbearably labored and ultimately nonfunctional. Much research, therefore, has gone
into understanding the mechanisms, influences, and tendencies of cancerous cells. A number of
variables have been shown to have a remarkable influence upon the developmental conditions
expressed by these cellular phenotypes, including the presence and concentration of specific
growth factors, extracellular matrix components and structure, as well as the physical structures
inherent to the cells themselves.
Despite progress in medical practices and technology, glioblastoma multiform continues
to present as one of the most malignant forms of cancerous cerebral tumors. Accounting for
approximately 17% of all primary brain tumor types, incidence rates range around 3.2 per
100,000 people per year in the United States and Europe1-3
. A rapid, infiltrative cellular
phenotype characterizes glioblastoma tumors, often leading to tumor necrosis and
uncontrollable vascular proliferation reflected through a malignant morphology. Glioblastoma
can present in one of two ways: as an initial high-grade lesion that is highly recognizable during
brain imagining, or through time-dependent development from a lower-grade precursor lesion4
.
As the disease progresses, glioblastoma masses increase in size and typically contain central
areas of tissue necrosis, surrounded by widespread, peritumoral vasogenic edema5,6
. The
prognosis for patients diagnosed with glioblastoma cancer types is dismal. Despite treatment
options, median survival times hoover around 12 to 15 months after diagnosis, with a five-year
survival rate of less than 5%7,5
.
Histologically defined by numerous multinucleated giant cells with various morphological
features, glioblastoma presents with numerous proliferative, migration, and varying other
physiological features. These morphological features have shown to be influenced by the
structure of the surrounding extracellular matrix (ECM), as well as by the presence of gradients

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imposed by nutrients and other compounds necessary for cell vitality8-17
. Little research,
however, has gone into studying the effects of diffusible gradients mediating the migration of
tumor masses. Thus, in our original application we proposed to evaluate the role of ECM
proteins on glioblastoma cell migration, proliferation and metastasis in response to diffusive
chemogradients.
RESULTS & DISCUSSION
A. Design & development of microfluidic device
The ECM microenvironment is characteristically difficult to model in vivo. To overcome
this challenge, we have introduced a microfluidic device able to integrate multiple laboratory
conditions upon a single chip, a few millimeters in size (Fig. 1A). This platform was fabricated
using a silicon wafer developed using photolithographic processes. The initial mold design was
created using 3D CAD design software (SolidWorks) and fabricated at Stanford University. A
finite element analysis and simulation packaged (COMSOL) software was used to properly
understand the rate of diffusion of our studied growth factors through the various concentrations
of collagen matrices before any actual laboratory experiments were performed. COMSOL
Multiphysics software offers a microfluidic module able to integrate every aspect of our
experimental design, including matrix and growth factor concentration, with the actual device
proposed for our studies. The module includes interfaces for studying laminar flow, the common
mechanism of movement for growth factor gradients through a microfluidic platform. A 3D CAD
file obtained from the design of the microfluidic device in SolidWorks was first uploaded into the
COMSOL microfluidic module. Parameters such as diffusion coefficients and concentration
were input and boundary conditions specifying the exact locations of the introduced gradients
were selected. All diffusion coefficients were calculated using the Einstein-Stokes equation.
Diffusion coefficients were found to increase with particle size but decrease with an increase in
collagen concentration (Fig. 1B). The concentration of the given growth factor had no impact on
the diffusion coefficient. Running the simulation software resulted in a color oriented depiction of
the growth factor gradient through the uploaded microfluidic device, as well as a 2D plot
charting the concentration over a specified time interval along the length of the injected channel.
B. Implementation of device for cancer cell culture
Microfluidic devices fashioned from the designed mold in our lab are made with
polydimethylsiloxane (PDMS), as this medium is easily fabricated, translucent in appearance,

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and offers little resistance to alterations in physical properties such as elasticity, gas
permeability, biological inertness, etc. A multitude of growth factors and cell-matrix interactions
influence the motility of cells within both healthy and diseased microenvironments. While many
studies have been done showing the role of the extracellular matrix on cell adhesion and
migration within tumor sites, the structural-mechanical properties underlying matrix porosity,
stiffness, etc. remain to be considered. In addition, the role of various growth factors and related
biomarkers in neuronal ECM is of equal value for investigation. The microfluidic devices
proposed by this study offers researchers the ability to establish steady-state concentration
gradients of specified growth factors. These growth factors include various concentrations of
vascular epidermal growth factor (VEGF) and epidermal growth factor (EGF), both of these
biomolecular cues having a predominant influence upon cell mobility, proliferation, etc.
Cell behavior in vitro is particularly influenced by the rich concentration of type I collagen
present within the extracellular environment. As such, collagen matrices were prepared at 1, 2,
and 3 mg/ml, as designated in the experimental design. Glioblastoma cells were cultured within
3D gels of different concentrations and compositions of collagen-1, and further introduced to
various concentrations of EGF and VEGF growth factors. Finally, the cell shape index within the
3D gel and the number of cells migrated, along with their distance and velocity were quantified
using image analysis at regular intervals over a 48 h time period.
Results found thus far have indicated that the composition of matrix along with the
introduction of increasing concentrations of growth factors have a direct influence upon the
mobility of cancerous cell types (Fig. 1C, D). Cellular phenotype (Fig. 3A, B) is significantly
affected, with increasing matrix stiffness and decreasing pore sizes lengthening cell bodies,
arguably by increasing the available binding sites for cell attachment. The number of cells
migrated into the scaffolds, including velocity (Fig. 3C) and distance moved (Fig. 2 A, B)
steadily increased with the amplification of growth factor concentration. Definite quantitative
relationships between concentration gradients, cell migration distance and cell surface marker
expression levels have been therefore been found. Further investigation into the role of cell-cell
and cell-matrix interactions is to be done to better understand various influences upon cancer
cell mobility and tumor formation. Results suggest that stable gradients of EGF and VEGF
implemented within a microfluidic platform should directly influence the behavior of cells, even
when cultured with differing cell phenotypes or matrix interactions influenced by introduced
scaffold components. Further research into the motility of glioblastoma cells can profoundly
impact the development of migration-target approaches seeking to treat both adult and pediatric
glioblastoma.

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C. Future directions:
The PI is in the process of preparing a manuscript for submission of the outcomes of this
research to a peer-reviewed journal. Much of the work accomplished has been done for thesis
research of a graduate student (Ms. Amanda Powell, graduate student in BME program) within
the PI’s laboratory. In the next few months, the PI is planning to submit a proposal to National
Science Foundation based on the outcomes of this work.
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Figure 1. (A) Schematic of the microfluidic device developed for the cancer cell chemotaxis
study. The device has designated chambers for cell seeding, separate gel-filling ports for
injecting collagen gel into the designated gel chambers, and separate growth factor loading
chamber to create diffusive gradient across the scaffold. (B) COMSOL snapshot of the diffusion
of VEGF chemogradients through the 2 mg/ml collagen scaffold at 24 h time point.
Quantification of the total number of cells migrated through the 1 mg/ml (C) and 2 mg/ml (D)
collagen scaffolds under VEGF and EGF gradients.

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Figure 2. Chemotaxis of glioblastoma cells within 1, 2 and 3 mg/ml collagen scaffolds is highly
dependent on the VEGF (A) and EGF (B) concentrations and their gradients through the
scaffolds.

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Figure 3. (A, B) The effect of various concentration gradients of VEGF (A) and EFG (B) on cell
shape index (CSI), when cells were cultured within 1, 2 and 3 mg/ml collagen scaffolds. The
average velocity of cells (C) migrating through 1, 2 and 3 mg/ml collagen scaffolds is highly
dependent on the concentration gradients of VEGF and EGF.