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.

Figure 1. : (i) force measurement equations, (ii) force measurement on a DBTRG cell deflecting a fiber

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

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.

References:

1. Holland EC. (2000), “Glioblastoma multiforme: The terminator,” Proceedings of the National Academy of Sciences :97:6242-4.

2. Louis D, Ohgaki H, Wiestler O, Cavenee W, Burger P, Jouvet A (2007), “The 2007 WHO Classification of Tumours of the Central Nervous System,” Acta Neuropathologica, 114:97-109.

3. Sykova E. (2000), “Plasticity of Extracellular Space,” The Neuronal Environment Brain Homeostasis in Health and Disease. Totowa, New Jersey 07512: Humana Press Inc.p. 57.

4. Charras GT. (2008), “A short history of blebbing,” Journal of Microscopy, 231:pp. 466-78

5. Kumar, Sanjay and Valerie M. Weaver, (2009), “Mechanics, malignancy, and metastasis: The force journey of a tumor cell,” Cancer Metastasis Rev, 28(1-2): pp, 113-127

6. Zheng, Xiaoyu R, and Xin Zhang. (2011), “Microsystems for cellular force measurement: a review,” J.Micromech. Microeng. 21 054003

7. Charras, G.and Ewa Paluch, (2008), “Blebs lead the way: how to migrate without lamellipodia”. Nature Reviews Molecular Cell Biology. 9, pp. 730-736.

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

9.  Nain, A. S.; Phillippi, J. A, Sitti, M.; Mackrell, J.; Campbell, P. G.; Amon, C. (2008), “Control of Cell Behavior by Aligned Micro/Nanofibrous Biomaterial Scaffolds Fabricated by Spinneret-based Tunable Engineered Parameters (STEP) Technique”, Small (Weinheim an der Bergstrasse, Germany) 4, 1153-9.

10. Ker, E. D. F.; Nain, A. S.; Weiss, L. E.; Wang, J.; Suhan, J.; Amon, C. H.; Campbell, P. G.(2011), “Bioprinting of Growth Factors onto Sub-micron Fiber-Based Scaffolds for the Regeneration of Musculoskeletal Interfaces,” Biomaterials, 32, 8097-107.

11.  Bakhru, S.; Nain, A. S.; Highley, C.; Wang, J.; Campbell, P.; Amon, C.; Zappe, S. (2011), “Direct and Cell Signaling-Based, Geometry-Induced Neuronal Differentiation of Neural Stem Cells,” Integrative biology : quantitative biosciences from nano to macro, 3, 1207-14.

12. Sharma P, Sheets K, and Nain AS,(2012), “The mechanistic influence of aligned nanofiber networks on cell shape, migration and blebbing dynamics of DBTRG cells”, Biomaterials, Submitted and under review