(717h) Nanofiber Based Cellular Protrusions of Cancerous Cells

Nanofiber based Cellular
Protrusions of Cancerous Cells

Brian Koons¹, Puja Sharma²,
Amrinder S. Nain^1,2

¹Mechanical
Engineering Department, Virginia Tech, Blacksburg, USA

²School
of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, USA

Introduction

As cells migrate and interact with their mechanical
environment of the extracellular matrix (ECM), they sense their surroundings
using cytoplasmic protrusions. Generally, these protrusions can be classified
as either lamellipodia or filopodia, where actin within the protrusion is in
the form of a broad branched sheet or thin long bundle, respectively [1]. Although
lamellipodia have been known to play a pivotal role on 2D flat substrates,
filopodia proves to be the dominant sensing mechanism in 3D microenvironments [2].
In cancerous cells, filopodia-like projections known as invadopodia are used to
break down the surrounding ECM and infiltrate the proximate blood vessels.
Current studies consist of using chemical gradients to force cancer cell
protrusions across a porous membrane. Here, alternatively, we demonstrate a biophysical
method using nanofibers to selectively position areas of contrasting structural
stiffness (N/m) in order to elicit cellular protrusions. Using the previously
reported STEP technique, non-electrospun suspended polymer nanofibers are
deposited on substrates with varying fiber diameters in aligned configurations
to create stiffness and geometric gradients sensed by the cell [3]. Cells interacting
with this system are seen to migrate on the stiff fibers while sending
protrusions towards and on the less stiff environment of nanofibers. Therefore,
this method when coupled with immunostaining, can provide an understanding of the surrounding biophysical
influence on actin polymerization and depolymerization. Use of this method provides a
protrusion-based comparison for the invasiveness and metastatic potential of
various cancer cell types including brain, breast, and prostate.

Materials
and Methods

Nanofibers were fabricated from polymer solutions
of polystyrene dissolved in xylene using various concentrations and molecular
weights. The fibers were suspended in a highly controlled and aligned fashion across
polystyrene frames via STEP technique to create a fibrous grid where crossing
fibers are perpendicular to one another. Vertical fibers averaged 2 µm in diameter while
horizontal fibers were 250 nm or 500 nm in diameter for each sample. Diameter
measurements were confirmed using scanning electron microscopy. Fusing fibers
at their intersections created unified fiber meshes with both fibers coexisting
on the same geometric plane. The fiber meshes were coated with ECM protein
fibronectin (2 µg/ml)
to promote cellular adhesion prior to cell seeding with Denver Based Tumor
Research Group (DBTRG-05MG) glioma cell line.
Time-lapse imaging and measurements were performed using Carl Zeiss microscope and Axiovision
software respectively. As cells initiated protrusions throughout the nanofiber
mesh, protrusion length (µm),
extension rate (µm/min),
and retraction rate (µm/min)
were measured using aforementioned software.

Results

Protrusion Behavior

DBTRG-05MG favors the 2 𝜇m
fibers, and mostly stayed wrapped around the 2 𝜇m
fibers due to high structural stiffness. Cells rarely migrated on less stiff
nanoscale fibers. Instead, the cells extended protrusions from the stiff
microfibers onto the less stiff, smaller diameter nanofibers, as shown in Figure
1. These protrusions were observed to be thin rod-like cytoplasmic
projections, and their occurrence was observed to depend on the fiber diameter
upon which they extended. Cells were observed to extend multiple protrusions in
opposing directions, sensing potential migratory pathways within distance of
contact.

Protrusion Analysis

The average protrusion length of 23 µm
was constant between both 250 nm and 500 nm fibers. In addition, the protrusion
extension rate of 3 µm/min
did not differ between diameters. However, the rate of protrusion retraction
for the larger diameter fibers (3.3 µm/min)
was slower than when compared to retraction rates on smaller diameter fibers
(4.2 µm/min) as
shown in Figure 1.

Figure 1.  (a) Cell
protrusion on large, 500 nm fiber, (b) bi-directional cell protrusion on
medium, 250 nm fiber,  (c) protrusion
extension and retraction rates between large and medium fiber diameters, and
(d) total length of protrusion compared between large and medium fibers.

Discussion

The study shows that biophysical characteristics of the extracellular
microenvironment can control cell attachment and migration preference. Preliminary
data illustrates that retraction can be regulated through the variance of fiber
diameter. However, protrusion length and extension rates were seen to be
constant on the diameters tested. Although the protrusions are sensing the
mechanical characteristics of the extracellular environment, they can also be
seen as migratory attempts to overcome the prohibitory stiffness gradient which
is keeping the cell migrating along the large, stiffer fiber. Their protrusion
extending potential can quantify the invasiveness and metastatic potential of cancerous
cells. Our future experiments are aimed at exploring protrusion dynamics for a
variety of cancer and normal cell lines on fibers of different diameters and
structural stiffness.

References

[1] Faix, Jan Breitsprecher,
Dennis Stradal, Theresia E
B Rottner, Klemens.
Filopodia: Complex models for simple rods. The international
journal of biochemistry & cell biology. 2009

[2] Albuschies, Jörg Vogel, Viola. The role of filopodia in the recognition of nanotopographies.
Scientific Reports. 2013

[3] Nain AS, Sitti M, Jacobson
A, Kowalewski T, Amon C. Dry Spinning Based Spinneret Based Tunable Engineered
Parameters (STEP) Technique for Controlled and Aligned Deposition of Polymeric
Nanofibers. Macromolecular Rapid Communications.
2009;30:1406-12.