Lung Micrometastases Display ECM Depletion and Softening While Macrometastases Are 30-Fold Stiffer and Enriched in Fibronectin

Narciso, Maria; Martínez, África Martos; Júnior, Constança; Díaz-Valdivia, Natalia; Ulldemolins, Anna; Berardi, Massimiliano; Neal, Kate; Navajas, Daniel; Farré, Ramón; Alcaraz, Jordi; Almendros, Isaac; Gavara, Núria

doi:10.3390/cancers15082404

Cited by 3 publications

(1 citation statement)

References 87 publications

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“…When these steps are performed, the output is often a large macroscopic gel, even when only a thin microscopic-scale gel surface is required or even preferred for cell culture and imaging. In our laboratory, we have developed optimized protocols for the cryosectioning of whole animal organs samples, using them for further cell culture, imaging, or mechanical characterization [18][19][20]. One of the key advantages is the ability to expand the number of samples that can be used from scarce tissue [18].…”

Section: Introductionmentioning

confidence: 99%

Cryosectioning of Hydrogels as a Reliable Approach to Increase Yield and Further Tune Mechanical Properties

Martínez-Blanco,

Noé,

Carreras-Vidal

et al. 2023

Gels

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Decellularized extracellular matrix (dECM) hydrogels have emerged as promising materials in tissue engineering. The steps to produce dECM hydrogels containing the bioactive epitopes found in the native matrix are often laborious, including the initial harvesting and decellularization of the animal organ. Furthermore, resulting hydrogels often exhibit weak mechanical properties that require the use of additional crosslinkers such as genipin to truly simulate the mechanical properties of the desired study tissue. In this work, we have developed a protocol to readily obtain tens of thin dECM hydrogel cryosections attached to a glass slide as support, to serve as scaffolds for two-dimensional (2D) or three-dimensional (3D) cell culture. Following extensive atomic force microscopy (AFM)-based mechanical characterization of dECM hydrogels crosslinked with increasing genipin concentrations (5 mM, 10 mM, and 20 mM), we provide detailed protocol recommendations for achieving dECM hydrogels of any biologically relevant stiffness. Given that our protocol requires hydrogel freezing, we also confirm that the approach taken can be further used to increase the mechanical properties of the scaffold in a controlled manner exhibiting twice the stiffness in highly crosslinked arrays. Finally, we explored the effect of ethanol-based short- and long-term sterilization on dECM hydrogels, showing that in some situations it may give rise to significant changes in hydrogel mechanical properties that need to be taken into account in experimental design. The hydrogel cryosections produced were shown to be biocompatible and support cell attachment and spreading for at least 72 h in culture. In brief, our proposed method may provide several advantages for tissue engineering: (1) easy availability and reduction in preparation time, (2) increase in the total hydrogel volume eventually used for experiments being able to obtain 15–22 slides from a 250 µL hydrogel) with a (3) reduction in scaffold variability (only a 17.5 ± 9.5% intraslide variability provided by the method), and (4) compatibility with live-cell imaging techniques or further cell characterization of cells.

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Section: Introductionmentioning

confidence: 99%

Cryosectioning of Hydrogels as a Reliable Approach to Increase Yield and Further Tune Mechanical Properties

Martínez-Blanco,

Noé,

Carreras-Vidal

et al. 2023

Gels

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Expression and Clinicopathological Significance of Extracellular Matrix Remodeling Markers in Esophageal Squamous Carcinoma

Fan,

Chen,

Liu

et al. 2024

Crit Rev Eukaryot Gene Expr

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Esophageal squamous cell carcinoma (ESCC) is a common malignancy of the gastrointestinal tract with a single therapeutic option and a lack of effective clinical therapeutic biomarkers. Extracellular matrix (ECM) remodeling plays a pro-carcinogenic role in a variety of malignancies, but its role in esophageal squamous carcinoma remains to be elucidated. In this study, we examined the expression levels of ECM remodeling markers in 71 pairs of esophageal squamous carcinoma tissues and normal tissues adjacent to the carcinoma using immunohistochemical staining, and analyzed their relationship with clinicopathological features and prognosis. The results suggested that extracellular matrix remodeling markers (integrin Î±V, fibronectin, MMP9) were abnormally highly expressed in esophageal squamous carcinoma tissues. There was a statistically significant difference between the positive expression of ECM remodeling and the TNM stage of esophageal squamous carcinoma, and there was no statistically significant correlation with age, gender and carcinoembryonic antigen expression, differentiation degree, T stage, and lymph node metastasis. Overall survival rate and overall survival time were significantly lower in patients with positive ECM remodeling expression, which was an independent risk factor for poor prognosisof esophageal squamous carcinoma.

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Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch

Ulldemolins,

Narciso,

Sanz-Fraile

et al. 2024

Front. Cell Dev. Biol.

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Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs).Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72 h.Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions.Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

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Lung Micrometastases Display ECM Depletion and Softening While Macrometastases Are 30-Fold Stiffer and Enriched in Fibronectin

Cited by 3 publications

References 87 publications

Cryosectioning of Hydrogels as a Reliable Approach to Increase Yield and Further Tune Mechanical Properties

Cryosectioning of Hydrogels as a Reliable Approach to Increase Yield and Further Tune Mechanical Properties

Expression and Clinicopathological Significance of Extracellular Matrix Remodeling Markers in Esophageal Squamous Carcinoma

Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch

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