3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK

Abstract

Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using twodimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK-and F-actin-dependent manner.Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.

Rheological Properties of Coordinated Physical Gelation and Chemical Crosslinking in Gelatin Methacryloyl (GelMA) Hydrogels

“…It is known that compressive stresses will prevent spheroid growth due to volume constraints, an effect observed with various spheroid and scaffold types. [ 81–84 ] We observed the most limited growth in porcine GelMA polymerized at 4 °C, where we also observed the scaffold radially shrinking over time. Acellular porcine GelMA with high DS polymerized at 4 °C in a well plate exhibited a 28.7 ± 2.1% decrease in scaffold diameter 3 days after crosslinking.…”

“…highlights that supplementing GelMA scaffolds with fibrous collagen is required for MDAMB231 spheroid invasion into scaffolds with storage moduli ( G ′) higher than the optimal window identified herein. [ 84 ] Spheroid invasion was observed in composite GelMA‐collagen scaffolds with storage moduli ( G ′) as high as 12 kPa. This suggests spheroid invasion may be possible in GelMA scaffolds with coordinated physical gelation and chemical crosslinks upon supplementing the matrix with fibrous support materials, such as collagen.…”

EMT‐Induced Cell‐Mechanical Changes Enhance Mitotic Rounding Strength

“…Indeed, mechanical confinement or external pressure have been shown to hamper cell proliferation in tumor spheroids. [ 14–18 ] Thus, it has been hypothesized that the actin cortex of cancer cells exhibits oncogenic adaptations that allow for ongoing mitotic rounding and division inside tumors. [ 19 ] In fact, it was shown that the human oncogene Ect2 contributes to mitotic rounding through RhoA activation [ 7,10 ] and that Ras overexpression promotes mitotic rounding.…”

Mechanical properties of cell- and microgel bead-laden oxidized alginate-gelatin hydrogels

“…PAAm microgel beads have shown the potential to mimic cells with respect to their diameter and elasticity, and to allow for a comparison between different mechanical assessment techniques, such as atomic force microscopy (AFM) and real-time deformability cytometry (RT-DC). 91 Due to their mechanical similarities to cells, PAAm beads have been used as stress sensors, [91][92][93] for the calibration of RT-DC measurements, 94 and to build 3D colloidal scaffolds with spatially differing mechanical layers for cell growth, migration and mechanosensitivity studies. 95 Here, we investigate the mechanical properties of hydrogels containing PAAm microgel beads and embryo mouse fibroblast (NIH/ 3T3) cells for small and large deformations under compression, tension, and torsional shear loadings.…”