Viscoelastic relaxation of collagen networks provides a self-generated directional cue during collective migration

Author's response

Clark Andrew and Ananyo Maitra shared

Open questions 

1.This is a very interesting work. My first question is what is the minimal cluster size capable of inducing the changes in the collagen network necessary for migration?

Thank you. That is indeed a great question and something we are currently looking into. It is not clear whether there is truly a threshold size for producing collagen gradients, but we do see a correlation between cell/cluster size and gradient strength (which we can approximate by taking the ratio between the maximum and minimum collagen densities across the cell). From our initial analysis, it seems that the gradient strength seems to be very sensitive to changes in cell/cluster size near the size of a single cell. For larger clusters, the gradient strength is less sensitive. This could hint at a sharp transition between formation of a gradient or no gradient that looks similar to a size threshold near the size of a single cell.

We find a very similar scaling when we analyze how cluster size scales with migration dynamics. Migration persistence is very sensitive to changes in size for smaller clusters and single cells, but not for larger clusters. That means that the transition from non-persistent to persistent migration occurs for very small clusters, just above the size of a single cell. This corresponds with the threshold-like behavior we observe for the formation of collagen gradients.

The model does not tell us exactly how cluster size should scale with persistence, though it does allow us to predict a size dependence. Being able to measure this scaling experimentally, it will be informative to incorporate this into the model in the future.

2.You discuss your findings in the context of tumour migration. Do you expect this might differ from collective migration in other contexts, such as infectious pathogens migrating collectively within tissues inside a host, or immune cells migrating in response to infection?

I think our study deals with a particular form of collective migration, where the cells migrate together as a single cohesive unit. This would be most similar to border cell migration in Drosophila and lateral line formation in Zebrafish. In these systems, I would also expect that viscoelastic relaxation of the local environment could also play a role in collective migration.

In the case of collective migration of pathogens, like bacterial swarming, or directed migration of immune cells, I could also imagine similar effects of local ECM remodeling. These systems are inherently more complex because individual cells can interact with each other, and perturbations of the local environment by one cell could affect its neighbors. Here I would draw an analogy with self-generated chemotactic gradients, in which groups of cells migrate directionally in the presence of uniform chemokine concentration (Tweedy et al, 2016 PLoS Biol.). There, a wave of cells migrates up a local chemokine gradient as the cells consume the chemokine (which further creates the gradient). I could imagine a similar mechanism of substrate deformation: a wave of migrating cells at the leading edge create a wave of high substrate perturbation just behind the leading edge, which further directs the cells to move forward. In the case of swarming bacteria, this could be a hydrodynamic effect in the surrounding medium, while in immune cells or perhaps epithelial cells, this could be a wave of high ECM density, similar to what we observe in our system. However, this may differ for cell migration under confinement or for adhesion-free migration modes, where migration can occur in the absence of interactions with ECM and without exerting high stresses on the substrate. It will be interesting in the future to better understand how viscoelastic properties of confined environments can influence migration.

3.You discuss chemotaxis as a separate phenomenon throughout your work. In a physiological situation, how would both phenomena combine (ie chemotaxis and collagen remodelling) to result in cell migration?

This is a really great question that our group and others are starting to look into. Since we have only looked at migration in random conditions, we do not yet know how substrate remodeling and chemotaxis function simultaneously. We could well imagine that the sort of dynamic ECM remodeling that we see could further enhance chemotaxis because it promotes persistent migration. We would also expect that ECM remodeling could permit chemotaxis with a “pulsed” chemokine gradient. If a gradient were formed transiently, that would be enough to break symmetry and start cell migration toward the gradient; if the chemokine gradient were then removed, we would expect the cells to continue migrating in the same direction. In this sense, the ECM remodeling mechanism could reduce the burden of making a chemokine gradient since just a pulse of chemokine, and not a sustained gradient, may be enough to stimulate cells to migrate in the right direction. For similar reasons, it may also make it more difficult for a group of cells to “turn” if the gradient changes direction, since the substrate deformation would push the cells to continue moving forward. We would expect this to result in a time lag or delay when changing directions.

4.Different tissues have a different composition in their parenchyma. In a hypothetical situation considering the same cell type (individual or cluster) migrating, do you expect there are organs where collagen network remodelling is more efficient/easier than others, and this influences cell behaviour (including metastasis)?

Another excellent question. Our results suggest that if collagen is too stiff or recovers too quickly, migration persistence is reduced. With less persistent migration, groups of cells tend to explore a smaller area since they spend their time migrating back and forth in small circles. From this, it would follow that tissues with more deformable stromal networks would be more permissive for migration and potentially metastasis. However, high stiffness in the pre-metastatic niche could also encourage cells to stop migrating and start growing, which would favor metastasis formation. The requirement for migration at a secondary site or the ability of metastases to grow depends on a number of factors and is likely very tissue specific. This gets back to the old “seed and soil” hypothesis, which states that in order for cancer cells (the “seeds”) to mestasize (or “grow”), they must be placed in the right microenvironment (the “soil”; Paget 1889 Lancet). It is becoming increasingly clear that the mechanics of the microenvironment must be considered alongside biochemistry and signaling, and I think our study offers some insight into the importance of substrate mechanics in cell migration and potentially metastasis.

5.You discuss in the model and experimentally, relaxation times. Considering a physiological situation of a combination of various cell clusters and individual cells migrating collectively, how would collagen network remodelling by one cluster influence migration of individual cells following this cluster, and within which time-scale?

I would expect this sort of mixed population to display different behaviors depending on the relative positions of the clusters and single cells. Although single cells cannot generate strong enough gradients to migrate persistently on their own, they are still able to respond to imposed gradients. Based on preliminary analysis using similar theoretical models of autophoretic motion, we would predict that single cells in front of large clusters (where front and back are defined with respect to the direction of motion of the cluster) would be persistently pushed forward by the cluster. In contrast, any single cell behind a large cluster would be kicked backward, as it would experience a collagen gradient in the opposite direction, and the cell would not be predicted to migrate persistently. These behaviors would all occur on the timescale of collagen relaxation, ~10 min. Numerical models and simulations are really handy for exploring these questions, as the behavior would be expected to be very different for different cellular arrangements, even though the basic interaction rules are the same.

Considering long-timescale interactions, we would expect to see a gradual stiffening of the collagen after many migration events. We observe in our cell removal assays that collagen density does not completely relax to levels of the background density, suggesting that some of the collagen reorganization is permanent. Over time, we would expect collagen density to accumulate and result in stiffening. This would mean that the first clusters to migrate across a region of collagen would experience a softer, more deformable network, while clusters that migrate later may experience a less deformable network and, as a result, migrate less persistently.