Inherent Mechanical Gradients In 3D Matrigel Cultures Influence Tumor Cell Behaviors

It is widely appreciated that cells can sense stiffness of their environment and alter their response accordingly [1]. To examine the influence of stiffness on cell behaviors, hydrogels with independent and varying mechanical properties are required. Investigators have recently begun to incorporate stiffness gradients in hydrogel systems using photo patterning and microfluidic platforms [2]. Whereas most of these methods provide well defined gradients and control over material properties, they are usually time intensive often requiring several steps to generate gradients. Additionally, most are designed to examine cell behaviors in 2D cultures. Here, we exploit edge effects in existing 3D culture systems (gels supported on rigid substrates) to investigate the influence of stiffness gradients on tumor cells using Matrigel as a model hydrogel material. As a model tumor system, we investigated glioblastoma multiforme (GBM), an astrocyte-derived brain cancer and one of the most deadly forms of human cancer affecting ~22500 individuals in the United States annually. GBMs are highly infiltrative and inevitably recur both locally and distantly within the brain; median survival times are extremely low (~12-15 months) [3].

Whereas the bulk hydrogel modulus does not vary, in regions close to the rigid support (i.e., glass), edge effects create an inherent gradient in mechanical stiffness that varies between that of the support and the hydrogel. A finite element model was employed to confirm the presence of varying mechanical environments in 3D cultures. To investigate GBM response to these environments, patient derived GBMs (OSU-2) were encapsulated in different concentrations of Matrigel (40, 55, 70, 85 % v/v). In particular, we examined cell morphology and spreading, intracellular morphology (actin organization) and migration capacity of OSU-2 cells at the lowest gel positions and highest gel positions. OSU-2 cells close to the bottom of the substrate showed highly elongated, bipolar morphologies and had statistically different cell areas compared with those more distant from the rigid support, which showed mostly rounded morphologies (with short processes in some cases). This was also reflected in actin organization, with actin filaments being highly organized and forming stress fibers in cells close to the substrate whereas those nearer to the gel surface displayed poorly defined actin architecture. OSU-2 cells also migrated at significantly higher speeds close to the bottom of substrate in a mesenchymal fashion displaying long processes compared to those nearer to the gel surface that migrated more slowly while displaying rounded or ellipsoid cell bodies with short processes. This system is, by far, one of the simplest systems to study the influence of stiffness gradients on the behavior of tumor cells in a single 3D hydrogel system and also provides insights as to how far cells can “sense” their microenvironments in 3D.

References:

[1] D. E. Discher et al., Science. 2005. 310; 1139-43.

[2] S.  Sant et al., The Canadian Journal of Chemical Engineering. 2010.88; 899-911.

[3] P. Y. Wen et al., N Engl J Med. 2008. 359 (5); 492-507.