The Mechanical Rigidity of the Extracellular Matrix Regulates the Structure, Motility, and Proliferation of Glioma Cells

Summary

Astrocytomas are tumors that originate in star-shaped brain cells. The World Health Organization classified Glioblastoma multiforme (GBM) with a grade 4 making it one of the most aggressive astrocytomas known. GBM’s aggressiveness is due to the ability of a single tumor cell to infiltrate the brain parenchyma even before diagnosis, preventing the surgical extraction of the tumor. Current research focuses on uncovering the factors that influence GBM cell structure, motility , and proliferation. This includes GBM’s interactions with the extra cellular matrix (ECM) (specifically ECM proteins fibronectin, laminin, and collagen) and the molecular components within the cell that respond to ECM cues (actin-myosin network and signaling molecules such as RhoA, RhoB, and NMMII expression).

In order to test the relationship between GBM cells and ECM, polyacrylamide gels of varying stiffnesses were synthesized (ranging from one order of magnitude below normal brain tissue to two orders of magnitude above). Cells from 4 diff GBM cell lines were plated on these gels.

Initial observation showed that decreasing substrate stiffness corresponded to a decrease in contact area of the cells. Cells on stiff substrates were well spread and had a well-developed network of actin stress fibers and discrete elongated vinculin positive focal adhesions while the cells on the softer substrates were more rounded, with fewer stress fibers present (As seen in the figure below).

Glass, the stiffest ECM was used as control. Cells plated on glass showed the same trend seen on the stiffer gels, which showed that cells’ adhesion-based cytoskeleton assembly was independent of the polymerization chemistry of the gels.

Timelapse imaging was used to observe cell migration on each of the different gels. As substrate rigidity was decreased, modes and speeds of cell migration changed, resulting in an overall decrease in migration speed (As seen in the figure below). Speeds were indistinguishable between the most rigid substrates and glass.

It was seen that ECM also affected tumor cell proliferation. Initial observation showed that cultures on stiff substrates reached confluency faster than cells on softer substrates. This observation was further investigated by performing a BrdU stain wherein cells incorporated BrdU into their DNA as they divided allowing the measurement of percentages of dividing cells as a function of ECM rigidity (As seen in the figure below). This confirmed the initial observation that stiffer substrates increase cell proliferation.

It was also hypothesized that NMMII and its upstream regulators were responsible for processing ECM cues. This was tested by inhibiting NMMII in these cells and observing cell behavior on the gels of differing stiffnesses. Previous experiments showed that a soft ECM decreased cell motility and proliferation. However soft ECM no longer impeded cell motility and proliferation on cells whose NMMII expression was suppressed. This shows that NMMII and its regulators are essential to the cell’s ability to receive and react to the ECM cues.

Significance

The paper showed a direct correlation between ECM rigidity and cell structure, motility, and proliferation. As substrate rigidity increased, cell adhesion, motility and proliferation were enhanced, effectively turning a group of GBM cells into a malignant tumor. Normal brain tissue corresponds to the rigidity of the compliant matrices tested in the paper, leading to the hypothesis that tumor cells were able to remodel the ECM, making it stiffer and hence favorable for cell adhesion, motility, and proliferation. This finding is significant because it leads to mechanobiologically-inspired therapeutics that focus on manipulating the ECM of GBM cells, possibly decreasing the rigidity surrounding tumor cells to reduce their growth and potential for damage. Furthermore, it was also shown that manual inhibition of NMMII or its upstream regulator ROCK via administered drugs decreased GBM's ability to respond to ECM cues, allowing the tumor to become malignant even on a compliant matrix. This proved that the NMMII signaling pathway is very important in sensing ECM rigidity and allowing the cells to respond accordingly. Understanding the molecular components responsible for the observable physiological change in these cells is important in being able to further manipulate cell behavior in the context of it’s microenvironment.

Critique

The paper does a good job of tying in experimental data with the initial hypotheses regarding mechanoregulation. Although the cells’ physiological responses were explained with respect to the substrates of differing stiffnesses, it might have been beneficial to test cell behavior on substrates of intermediate stiffnesses to the three that were used to confirm that the existence of the trends explained. This would also enable the depiction of the data on a plot, in the form of a curve that would help extrapolate the results to other applications or hypotheses being tested.

Although the molecular components within a cell responsible for its’ detection and response to ECM cues were discussed, further experimentation on the specific genes responsible for the activation/inhibition of the small molecules mentioned would have made this data stronger. For example, performing simple qPCR experiments to quantify levels of gene expression responsible for the interaction between the cells and ECM, and the physiological changes seen, would have provided quantitative information to back up the qualitative observations, in effect strengthening the conclusions made.