0.1 kilopascal difference for mechanophenotyping

Abstract

Current knowledge understands the mesenchymal cell invasion in a 3D matrix as a combined process of cell-to-matrix adhesion based cell migration and matrix remodeling. Excluding cell invasion stimulated by cytokines and chemokines, the basal cell invasion itself is a complicated process that can be regulated by matrix ligand type, density, geometry, and stiffness, etc. Understanding such a complicated biological process requires delicate dissections into simplified model studies by altering only one or two elements at a time. Past cell motility studies focusing on matrix stiffness have revealed that a stiffer matrix promotes 2D X-Y axis lateral cell motility. Here, we comment on two recent studies that report, instead of stiffer matrix, a softer matrix promotes matrix proteolysis and the formation of invadosome-like protrusions (ILPs) along the 3D Z axis. These studies also reveal that soft matrix precisely regulates such ILPs formation in the stiffness scale range of 0.1 kilopascal in normal cells. In contrast, malignant cells such as cancer cells can form ILPs in response to a much wider range of matrix stiffness. Further, different cancer cells respond to their own favorable range of matrix stiffness to spontaneously form ILPs. Thus, we hereby propose the idea of utilizing the matrix stiffness to precisely regulate ILP formation as a mechanophenotyping tool for cancer metastasis prediction and pathological diagnosis.

“Car on Mud”: Switching from 2D X-Y Axis Lateral Migration to 3D Z Axis Invasion

In vivo single cell motility can be categorized into at least four types: (1) 2D cell migration on a 2D surface, such as cells migrate on a blood or lymphatic vessel wall^1-3; (2) 3D cell invasion through a 3D extracellular matrix. For example, cells invade through soft matrices in the skin or a solid tumor microenvironment^4-6; (3) 1D cell migration and invasion through confined spaces. Recent intravital in vivo imaging studies have revealed that cells can migrate through very narrow pre-existing tissue tracks.^7,8 Ex vivo studies have also reported that cells can rapidly migrate through confined spaces that are as narrow as 3µm^9,10; (4) 2.5D cell invasion. For example, when a cell decides to invade through the blood vessel wall, it stops its 2D lateral motility on the vessel wall and starts to invade along the 3D Z axis that is perpendicular to the 2D vessel wall surface.¹¹ The study we have performed on soft matrix is relatively similar to such 2.5D cell invasion in vivo, when cells stop 2D X-Y axis lateral motility and start to secrete MMPs and form ILPs to invade along the 3D Z axis.¹²

Cell X-Y axis lateral migration on a 2D rigid surface relies on the traction forces between the cell and the surface, which can provide enough friction for the cell to adhere, grab, and pull the adhesion ligands on the surface so as to achieve its forward motion.¹³ However, the adhesion ligands on a very soft surface are not stable. Thus, when a cell pulls these adhesion ligands, they move into the cell pulling direction and so fail to provide sufficient traction forces and friction for a cell to move forward.^14,15 Hence, cells display diminished X-Y axis lateral motility on a very soft matrix. However, we found cells do not become quiescent. We observed that on a very soft matrix whose stiffness (~0.2 kPa) is comparable to in vivo soft tissues, primary fibroblasts secrete and activate abundant MMPs, and meanwhile, they spontaneously form ILPs to degrade the substrate matrix in situ.^12,16 Such a finding suggests that soft matrix is capable of promoting spontaneous cellular motility switches from 2D X-Y axis lateral migration to 3D Z axis invasion. This phenomenon is comparable to a car on mud when a vehicle loses X-Y lateral frictions and starts to sink downward along the Z axis.

Soft Matrix Precisely Regulates Cellular Architecture

Interestingly, for primary human fibroblasts and normal endothelial cells on rigid surfaces with stiffness from gigapascal (glass, plastic, etc.) all the way down to ~20 kPa, cellular adhesion architectures including adherens junctions, focal adhesions and actin stress fibers display almost no difference. Strikingly, on relatively soft surfaces with stiffness from ~20 kPa down to 0.1 kPa, a slight matrix stiffness decrease significantly alters these cellular adhesion architectures.¹² For example, the size of focal adhesions decreases concurrently with matrix stiffness decreases from ~20 kPa down to 0.4 kPa. And surprisingly, these cells completely lose focal adhesions but form prominent ILPs instead on softer surfaces of 0.2 kPa stiffness, which is only a 0.2 kPa stiffness decrease from 0.4 kPa. Coincidently, the basal stiffness of soft tissues such as lymph nodes, liver, and lung ranges from ~0.1 to ~20 kPa.^14,17,18 Thus for normal cells, such ex vivo findings suggest soft tissues in vivo, but not stiffer tissues, can precisely and profoundly regulates cellular architecture for cell adhesion and invasion by a slight stiffness change in the scale range of 0.1 kPa.

Soft Matrix Induced ILP Formation for Mechanophenotyping in Cancer Diagnosis and Cancer Metastasis Prediction

Distinctively, in comparison to normal cells, malignant cells, such as cancer cells and sarcoma cells, spontaneously form ILPs in response to a much wider range of matrix stiffness. It seems that an individual type of cancer cell has its own signature stiffness spectrum for spontaneous ILP formation.¹² And further, such ILP formation diminishes when matrix stiffness increases. Hence, each type of cancer cell has its own fingerprint ILP formation curve if we plot number of cells forming ILPs against an axis of stiffness spectrum in a 2D quantification chart.¹² Current knowledge suggests cell invasion positively correlates with ILP formation.^19-22 Thus, such mechanophenotyping of soft matrix induced ILP formation could be applied to identifying different types of cancer cells that have distinct matrix invasion and metastatic capabilities.

Further, different types of cancer cells have noticeable differences in migration speed in response to matrices with various stiffness.^23-26 Hence, to magnify the resolution of such quantitative mechanophenotyping to identify different cancer cells with distinct metastatic capabilities, a 3D quantification chart with an X-axis of matrix stiffness, a Y-axis of percentage of cells forming ILPs and a Z-axis of cell migration speed could be plotted for such a purpose (Fig. 1).

Figure 1. Cell invasion mechanophenotyping. Percentage of cells forming ILPs in response to varying matrix stiffness and cell migration speed changes in response to varying matrix stiffness are plotted in a 3D quantification chart. Color balls represent individual cancer cells that have distinct invasion capabilities as determined by ILP formation and cell migration speed changes in response to matrices with various stiffness.

In conclusion, soft matrix precisely and differentially regulates cellular architecture for invasion in both primary normal cells and malignant cancer cells in the scale range of 0.1 kPa. We propose that such dramatic cellular architecture alteration in response to tiny changes of matrix stiffness could be applied as a cancer mechanophenotyping tool for cancer pathological diagnosis and cancer metastasis prediction.

Glossary

Abbreviations:

ILPs

invadosome-like protrusions

kPa

kilopascal

gPa

gigapascal

1D

one-dimensional

2D

two-dimensional

3D

three-dimensional

10.4161/bioa.29175

Gu Z, Liu F, Tonkova EA, Lee SY, Tschumperlin DJ, Brenner MB. Soft matrix is a natural stimulator for cellular invasiveness. Mol Biol Cell. 2014;25:457–69. doi: 10.1091/mbc.E13-05-0260.

Yu CH, Rafiq NB, Krishnasamy A, Hartman KL, Jones GE, Bershadsky AD, Sheetz MP. Integrin-matrix clusters form podosome-like adhesions in the absence of traction forces. Cell Rep. 2013;5:1456–68. doi: 10.1016/j.celrep.2013.10.040.