(781g) Single Cell Force Measurements of Glioblastoma Multiforme Using Aligned Nanofiber Networks

Single cell force measurements of Glioblastoma
Multiforme using aligned nanofiber networks

Puja Sharma¹,
Brian Koons², Tim O'Brien², Amrinder Singh Nain^{1, 2}

¹School of
Biomedical Engineering and Science, ²Mechanical Engineering

Virginia
Tech, Blacksburg, VA, USA

Introduction: With an average life expectancy of
15 months, glioblastoma multiforme (GBM) is the most invasive brain tumor of
glial origin in humans [1, 2]. About one fourth of the central nervous system
(CNS) volume is occupied by the extracellular space containing metabolites,
hormones and proteins secreted by the neurons and glia. Tumor cells use their
immediate extra cellular matrix (ECM) protein fibers (tens to hundreds of
nanometers in diameter) to metastasize within and out of the CNS [3]. A
phenomenon that has been associated with tumor migration, resistance to cell lysis and apoptosis is plasma membrane blebbing. Blebs are
short lived (<1 minute) circular (<15μm in
diameter) extensions that expand off the plasma membrane, and retract to the
initial point of expansion. Although it has been associated with migration, multidrug
resistance in cancer, changes in nuclear shapes and mitotic disturbance, its
specific functions are yet to be recognized. Given the recent association of
blebbing and cancer, the study of cancerous cell membrane blebbing requires
detailed exploration of the biophysical and biochemical cues linking this
phenomenon [4].

Glioma
cells remodel their immediate ECM to facilitate proliferation and invasion. In
doing so, they both endure and apply mechanical forces on their
microenvironment. This interplay of forces exerted by the ECM to the cell and
compensatory forces exerted by tumor cells to their environment plays a
critical role in motility, proliferation and tumorigenesis
[5]. Individual cell migratory forces are extremely challenging to measure as
they are in the pico to tens of nanoNewton range. Existing force analysis platforms to
measure single cell response to external mechanical stimuli either apply
external forces to the cell (Atomic Force Microscopy, Micropipette aspiration,
Optical/Magnetic tweezers) or utilize platforms that do not resemble the native
fibrous ECM (flat glass, micropillar, hydrogels) [6].
In this study, we present a previously described unique polymer nanofibrous
system of fused STEP fibers [8-11], modeled as mechanical springs upon which
cells migrate, resulting in individual fiber (spring) deflections, which are
used to calculate the migratory forces using Euler beam mechanics in the
elastic limit (Fig. 1). The key advantages of this approach are: (a) scaffolds mimic
the native ECM, (b) nanofibers of varying beam stiffness (N/m) can be used to
interrogate cellular behavior, and (c) fused fiber networks provide clamped
boundary conditions reducing complexity and allowing force estimation using
standard Euler mechanics.

Using
this platform [8-11], the forces exerted by a glioblastoma cell line
established from a female patient (Denver Based Tumor Research Group, DBTRG) as
a function of cell stretch area, bleb size and bleb count has been measured. Blebbing
has been considered as an alternate motility mechanism to the much studied
lamellipodia-based migration in two dimensional flat surfaces [7]. This
platform serves as a unique 3D setting that allows the analysis of force
exerted by individual blebbing glioma cells in motion. In doing so, a
quantitative measure of the magnitude of force exerted by blebbing cells (possibly
in amoeboid migration) is achieved. This could add a new dimension to the
understanding cell-ECM two way forces which modulate important aspects of
cancer progression such as migration, proliferation, and invasiveness.  

Materials and
Methods: Polymer
solutions of 7% and 10% (w/w) polystyrene dissolved in xylene were used to
manufacture highly aligned nanofiber (average diameter: 600nm, length: 6mm) grids
using previously described STEP technique [8-11]. Intersecting fibers were
fused and coated with fibronectin, to facilitate cell adhesion. After 5 hours
of seeding, time lapse video images of cells were taken using Zeiss microscope
with incubating capacity. AxioVision® software was
used to measure fiber length, cell size, bleb occurrence (counts), bleb size and fiber deflections. Obvious non-blebbing cells
were ignored during data collection. Force measurements were done using beam
mechanics theory (Fig.1). Cells were also fixed and immunostained
for nucleus (DAPI), actin (Phalloidin), and focal adhesions (Paxillin) (Fig.
3).

Figure 2. Force measurements of DBTRG as a function of (i) cell stretch area, (ii) bleb count and (iii) bleb size. As cell increased in stretch area, force exerted by the cell also increased. On the contrary, as bleb count and size increased, force exerted by the cell decreased.
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Figure 1. : (i) force measurement equations, (ii) force measurement on a DBTRG cell deflecting a fiber
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Figure 3. Immunostaining of DBTRG cells, blue (Nucleus, DAPI), red (actin, Phalloidin), green (Paxillin, Alexa Fluor), (i) blebbing cell, (ii) stretched cell showing prominent actin structures
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Results and
Discussion:
DBTRG cells attached, and pulled onto fibers as they migrated along nanofiber
grids (Fig. 1). Fiber lengths and deflection measurements allowed the
calculation of forces exerted by cells.  Deflections were monitored over time giving
force values from 10 nN to 150 nN.
Moreover, cell stretch area was proportional to force exerted by the cell. Both
bleb size and count were inversely proportional to force exerted by the cell
(Fig. 2).

Cytoskeleton
dynamics, especially actin plays a key role in modulating these forces. A
possible explanation of this behavior is that as cells stretch, actin assembly becomes
more pronounced facilitating the ability of the cells to exert higher forces
(Fig. 3 (ii)). Previously, our group has demonstrated a reduction in blebbing
as cells stretched in area [12].  Blebbing
cells therefore display less prominent actin assembly limiting the amount of
force the cells can exert on their microenvironment (Fig. 3 (i)). This data suggests that stretched cells play a
prominent role in exerting forces to their microenvironment possibility contributing
to the majority of cell to microenvironment forces.  

Conclusion: In this study, a quantitative
measure of the impact of cell stretch area and blebbing on force exerted by the
cell is achieved. Results show that the forces exerted by cells on their
environment decrease as cells start blebbing. Thus, we have shown that blebbing
decreases the ability of the cells to exert force on its microenvironment. This
phenomenon, to the best of our knowledge, has not been reported before. The
results also show that stretched cells exert higher forces on their environment
suggesting that the stretched cells may play a dominant role in exerting the required
compensatory forces against its stiffer microenvironment. As compensatory
forces exerted by tumor cells to their native environment play a crucial role
in motility, proliferation and tumorigenesis, the
results from this study could be used to better understand the mechanical
influence component of cell-ECM interaction in cancer progression.

This
study of individual cell forces as a function of cell stretch area and blebbing
contributes to the biophysical understanding of GBM cell behavior. This
platform better mimics native ECM and is a better model to study cellular
behavior. With the knowledge of forces exerted by glioma cells, a mechanistic
attribute of metastasis can be obtained. In future, this technology could be used
to study the remodeling mechanisms of the ECM, to characterize different stages
of invasiveness of GBM, or to study the effectiveness of different anticancer
agents, ultimately leading to better prognosis of GBM.

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Submitted and under review