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Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

ABSTRACT: Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

SUBMITTER: Conway JRW

PROVIDER: S-EPMC10614832 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

Conway James R W JRW Isomursu Aleksi A Follain Gautier G Härmä Ville V Jou-Ollé Eva E Pasquier Nicolas N Välimäki Eetu P O EPO Rantala Juha K JK Ivaska Johanna J

Proceedings of the National Academy of Sciences of the United States of America 20231016 43

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and ...[more]

PMID: 37844244

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Project description:Antifibrotic drug screening requires evaluating the inhibitory effects of drug candidates on fibrotic cells while minimizing any adverse effects on normal cells. It is challenging to create organ-specific vascularized organoids that accurately model fibrotic and normal tissues for drug screening. Our previous studies have established methods for culturing primary microvessels and epithelial cells from adult tissues. In this proof-of-concept study, we used rats as a model organism to create a two-dimensional vascularized liver organoid model that comprised primary microvessels, epithelia, and stellate cells from adult livers. To provide appropriate substrates for cell culture, we engineered ECMs with defined stiffness to mimic the different stages of fibrotic tissues and normal tissues. We examined the effects of two TGFβ signaling inhibitors, A83-01 and pirfenidone, on the vascularized liver organoids on the stiff and soft ECMs. We found that A83-01 inhibited fibrotic markers while promoting epithelial genes of hepatocytes and cholangiocytes. However, it inhibited microvascular genes on soft ECM, indicating a detrimental effect on normal tissues. Furthermore, A83-01 significantly promoted the expression of markers of stem cells and cancers, increasing the potential risk of it being a carcinogen. In contrast, pirfenidone, an FDA-approved compound for antifibrotic treatments, did not significantly affect all the genes examined on soft ECM. Although pirfenidone had minor effects on most genes, it did reduce the expression of collagens, the major components of fibrotic tissues. These results explain why pirfenidone can slow fibrosis progression with minor side effects in clinical trials. In conclusion, our study presents a method for creating vascularized liver organoids that can accurately mimic fibrotic and normal tissues for drug screening. Our findings provide valuable insights into the potential risks and benefits of using A83-01 and pirfenidone as antifibrotic drugs. This method can be applied to other organs to create organ-specific vascularized organoids for drug development.

Dataset Information

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

Publications

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

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