Dataset Information

Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile.

ABSTRACT: Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)-an actin-binding protein-levels in melanoma cell lines and GSN's importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin's BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN's influence on parameters associated with cells' ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin's BM.

SUBMITTER: Mazurkiewicz E

PROVIDER: S-EPMC8394273 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile.

Mazurkiewicz Ewa E Makowiecka Aleksandra A Mrówczyńska Ewa E Kopernyk Iryna I Nowak Dorota D Mazur Antonina Joanna AJ

Cells 20210721 8

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)-an actin-binding protein-levels in melanoma cell lines and GSN's importance for migration of A375 cells. Here we investigate whether melanoma ...[more]

PMID: 34440617

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Project description:Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. Impact statement Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Project description:IntroductionMacrophage phenotypes have been linked to progression and prognosis of cutaneous melanoma. However, the association between Warburg effect in A375 melanoma and macrophages polarization, as well as the underlying mechanisms, remains less well documented.ObjectiveThe present study aimed to investigate the effect of lactate derived from A375 melanoma on macrophage polarization, melanoma phenotype responses and the underlying mechanisms.MethodsFlow cytometry was performed to evaluate the expression of M1 and M2 markers, cell cycle and apoptosis. Levels of transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α) were determined with enzyme-linked immunosorbent assay (ELISA) kit. Proliferation and invasion were assessed by CCK8 and transwell assays, respectively. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed using an XF96 extracellular flux analyzer. Protein expressions were determined by Western blotting.ResultsOur results revealed that melanoma A375 conditioned medium (A375-CM) induced peripheral blood mononuclear cells (PBMCs) to polarize toward anti-inflammatory M2 macrophages. M2 markers CD206 and ARG1 expression increased, as did TGF-β secretion. Conversely, M1 marker CD68 expression decreased. Furthermore, hypoxia promoted macrophage M2 polarization induced by A375-CM. Elevated lactate level in PIG1-conditioned medium (PIG1-CM) induced M2 polarization, whereas the lactate transport inhibitor AZD3965 suppressed this effect in PBMCs cultured with A375-CM. Additionally, lactate derived from melanoma regulated M1/M2 polarization by the tricarboxylic acid (TCA) cycle instead of glycolysis. Significantly, polarized macrophages altered melanoma phenotypes including proliferation, clone formation, cell cycle, apoptosis, migration and invasion via TCA cycle and TGF-β.ConclusionOur data collectively demonstrate that lactate derived from melanoma facilitates polarization of M2 macrophages, which subsequently leads to modifications in melanoma phenotypes via TCA cycle and TGF-β signaling.

Dataset Information

Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile.

Publications

Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile.

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