In situ modulation of intestinal organoid epithelial curvature through photoinduced viscoelasticity directs crypt morphogenesis.

Spatiotemporally coordinated transformations in epithelial curvature are necessary to generate crypt-villus structures during intestinal development. However, the temporal regulation of mechanotransduction pathways that drive crypt morphogenesis remains understudied. Intestinal organoids have proven useful to study crypt morphogenesis in vitro, yet the reliance on static culture scaffolds limits the ability to assess the temporal effects of changing curvature. Here, a photoinduced hydrogel cross-link exchange reaction is used to spatiotemporally alter epithelial curvature and study how dynamic changes in curvature influence mechanotransduction pathways to instruct crypt morphogenesis. Photopatterned curvature increased membrane tension and depolarization, which was required for subsequent nuclear localization of yes-associated protein 1 (YAP) observed 24 hours following curvature change. Curvature-directed crypt morphogenesis only occurred following a delay in the induction of differentiation that coincided with the delay in spatially restricted YAP localization, indicating that dynamic changes in curvature initiate epithelial curvature-dependent mechanotransduction pathways that temporally regulate crypt morphogenesis.

Quantifying stiffness and forces of tumor colonies and embryos using a magnetic microrobot.

Stiffness and forces are two fundamental quantities essential to living cells and tissues. However, it has been a challenge to quantify both 3D traction forces and stiffness (or modulus) using the same probe in vivo. Here, we describe an approach that overcomes this challenge by creating a magnetic microrobot probe with controllable functionality. Biocompatible ferromagnetic cobalt-platinum microcrosses were fabricated, and each microcross (about 30 micrometers) was trapped inside an arginine-glycine-apartic acid-conjugated stiff poly(ethylene glycol) (PEG) round microgel (about 50 micrometers) using a microfluidic device. The stiff magnetic microrobot was seeded inside a cell colony and acted as a stiffness probe by rigidly rotating in response to an oscillatory magnetic field. Then, brief episodes of ultraviolet light exposure were applied to dynamically photodegrade and soften the fluorescent nanoparticle-embedded PEG microgel, whose deformation and 3D traction forces were quantified. Using the microrobot probe, we show that malignant tumor-repopulating cell colonies altered their modulus but not traction forces in response to different 3D substrate elasticities. Stiffness and 3D traction forces were measured, and both normal and shear traction force oscillations were observed in zebrafish embryos from blastula to gastrula. Mouse embryos generated larger tensile and compressive traction force oscillations than shear traction force oscillations during blastocyst. The microrobot probe with controllable functionality via magnetic fields could potentially be useful for studying the mechanoregulation of cells, tissues, and embryos.

Synthetic Retinoid Kills Drug-Resistant Cancer Stem Cells via Inducing RARγ-Translocation-Mediated Tension Reduction and Chromatin Decondensation.

A recently developed synthetic retinoid abrogates proliferation and induces apoptosis of drug-resistant malignant-cancer-stem-cell-like cells. However, the underlying mechanisms of how the synthetic retinoid induces cancer-stem-cell-like cell tumor-repopulating cell (TRC) apoptosis are elusive. Here, it is shown that although the retinoid and conventional anticancer drugs cisplatin, all-trans retinoic acid, and tazarotene all inhibit cytoskeletal tension and decondense chromatin prior to inducing TRC apoptosis, half-maximal inhibitory concentration of the retinoid is 20-fold lower than those anticancer drugs. The synthetic retinoid induces retinoic acid receptor gamma (RARγ) translocation from the nucleus to the cytoplasm, leading to reduced RARγ binding to Cdc42 promoter and Cdc42 downregulation, which decreases filamentous-actin (F-actin) and inhibits cytoskeletal tension. Elevating F-actin or upregulating histone 3 lysine 9 trimethylation decreases retinoid-induced DNA damage and apoptosis of TRCs. The combinatorial treatment with a chromatin decondensation molecule and the retinoid inhibits tumor metastasis in mice more effectively than the synthetic retinoid alone. These findings suggest a strategy of lowering cell tension and decondensing chromatin to enhance DNA damage to abrogate metastasis of cancer-stem-cell-like cells with high efficacy.

Microtissue Geometry and Cell-Generated Forces Drive Patterning of Liver Progenitor Cell Differentiation in 3D.

3D microenvironments provide a unique opportunity to investigate the impact of intrinsic mechanical signaling on progenitor cell differentiation. Using a hydrogel-based microwell platform, arrays of 3D, multicellular microtissues in constrained geometries, including toroids and cylinders are produced. These generated distinct mechanical profiles to investigate the impact of geometry and stress on early liver progenitor cell fate using a model liver development system. Image segmentation allows the tracking of individual cell fate and the characterization of distinct patterning of hepatocytic makers to the outer shell of the microtissues, and the exclusion from the inner diameter surface of the toroids. Biliary markers are distributed throughout the interior regions of micropatterned tissues and are increased in toroidal tissues when compared with those in cylindrical tissues. Finite element models of predicted stress distributions, combined with mechanical measurements, demonstrates that intercellular tension correlates with increased hepatocytic fate, while compression correlates with decreased hepatocytic and increased biliary fate. This system, which integrates microfabrication, imaging, mechanical modeling, and quantitative analysis, demonstrates how microtissue geometry can drive patterning of mechanical stresses that regulate cell differentiation trajectories. This approach may serve as a platform for further investigation of signaling mechanisms in the liver and other developmental systems.

Force-induced gene up-regulation does not follow the weak power law but depends on H3K9 demethylation.

Mechanical forces play important roles in development, physiology, and diseases, but how force is transduced into gene transcription remains elusive. Here, we show that transcription of transgene DHFR or endogenous genes egr-1 and Cav1 is rapidly up-regulated in response to cyclic forces applied via integrins at low frequencies but not at 100 Hz. Gene up-regulation does not follow the weak power law with force frequency. Force-induced transcription up-regulation at the nuclear interior is associated with demethylation of histone H3 lysine-9 trimethylation (H3K9me3), whereas no transcription up-regulation near the nuclear periphery is associated with H3K9me3 that inhibits Pol II recruitment to the promoter site. H3K9me3 demethylation induces Pol II recruitment and increases force-induced transcription of egr-1 and Cav1 at the nuclear interior and activates mechano-nonresponsive gene FKBP5 near the nuclear periphery, whereas H3K9me3 hypermethylation has opposite effects. Our findings demonstrate that rapid up-regulation of endogenous mechanoresponsive genes depends on H3K9me3 demethylation.

Quantifying compressive forces between living cell layers and within tissues using elastic round microgels.

Increasing evidence shows that mechanical stresses are critical in regulating cell functions, fate, and diseases. However, no methods exist that can quantify isotropic compressive stresses. Here we describe fluorescent nanoparticle-labeled, monodisperse elastic microspheres made of Arg-Gly-Asp-conjugated alginate hydrogels (elastic round microgels, ERMGs). We generate 3D displacements and calculate strains and tractions exerted on an ERMG. Average compressive tractions on an ERMG are 570 Pa within cell layers and 360 Pa in tumor-repopulating cell (TRC) colonies grown in 400-Pa matrices. 3D compressive tractions on a 1.4-kPa ERMG are applied by surrounding cells via endogenous actomyosin forces but not via mature focal adhesions. Compressive stresses are substantially heterogeneous on ERMGs within a uniform cell colony and do not increase with TRC colony sizes. Early-stage zebrafish embryos generate spatial and temporal differences in local normal and shear stresses. This ERMG method could be useful for quantifying stresses in vitro and in vivo.