A Fluorescent Gelatin Degradation Assay to Study Melanoma Breakdown of Extracellular Matrix

Mazurkiewicz, Ewa; Mrówczyńska, Ewa; Simiczyjew, Aleksandra; Nowak, Dorota; Mazur, Antonina Joanna

doi:10.1007/978-1-0716-1205-7_3

Cited by 6 publications

(9 citation statements)

References 21 publications

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“…Notably, in our degradation assays, sometimes we only observed shadows or lower intensity compared with the rest of the green gelatin in gelatin matrix instead of black holes, which is consistent with previous studies [ 39 , 41 , 42 ]. Mazurkiewicz E et al [ 41 ] pointed out that this phenomenon could be an incomplete degradation of gelatin in the three dimension, leading to degraded areas not shown as black hole but only seem to be less intense than the rest of the green FITC-gelatin layer. What is more, when aggressive degradation was seen in some certain cell types including MES-SA, pool-like clear area might be seen in gelatin matrix [ 40 , 43 ].…”

Section: Resultssupporting

confidence: 92%

TEM1 up-regulates MMP-2 and promotes ECM remodeling for facilitating invasion and migration of uterine sarcoma

Sun

Shen

et al. 2023

Discov Onc

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Objectives To explore the correlation between tumor endothelial marker 1 (TEM1) and matrix metalloproteinase 2 (MMP-2) in uterine sarcoma and their roles in the progression of uterine sarcoma. Methods Uterine leiomyosarcoma (uLMS, n = 25) and uterine leiomyoma (n = 25) specimens were collected from a total of 50 patients. Immunohistochemistry assay was conducted to determine the expression of TEM1, MMP-2 and MMP-9. TEM1 over expression (hTEM1) and low expression (shRNA-TEM1) MES-SA cell lines were established as in vitro uterine sarcoma models. MMP-2 mRNA, protein expression and enzymatic activity were verified using qPCR, Western blot and gelatin zymography respectively. MMP-2 expression was downregulated using MMP-2 siRNA in hTEM1 MES-SA cells to better study the role of MMP-2. The invasive and migratory capacities of hTEM1, shRNA-TEM1, and hTEM1 treated with MMP-2 siRNA MES-SA cells were determined using transwell assays. Extracellular matrix (ECM) remodeling mediated by TEM1 was examined using cell-ECM adhesion and fluorescent gelatin-ECM degradation assays. The immunofluorescence of F-actin was examined to analyze the formation of invadopodia. Subcutaneous and intraperitoneal xenografts were established to validate the role of TEM1 in promoting uterine sarcoma metastasis. Results TEM1 and MMP-2 were expressed in 92% (n = 23) and 88% (n = 22) of uterine leiomyosarcoma specimens, respectively. Both TEM1 and MMP-2 were highly expressed in 100% (n = 17) of high stage (III-IV) uterine leiomyosarcoma specimens. In addition, TEM1 expression was positively correlated with MMP-2 expression in uterine leiomyosarcoma. The successful establishment of in vitro uterine sarcoma models was confirmed with qPCR and Western blotting tests. TEM1 promoted the invasion and metastasis of uterine sarcoma in vivo and in vitro. MMP-2 expression and activity were up-regulated in hTEM1 cells but down-regulated in shRNA-TEM1 cells. Importantly, MMP-2 knockdown impaired the invasive and migratory capacity of hTEM1 cells. TEM1 promoted ECM remodeling by increasing cell-ECM adhesion and ECM degradation. TEM1 overexpression also induced the formation of invadopodia. Conclusion TEM1 was co-expressed and positively correlated with MMP-2 in uterine leiomyosarcoma specimens. In addition, both TEM1 and MMP-2 were associated with tumor development. TEM1 promoted uterine sarcoma progression by regulating MMP-2 activity and ECM remodeling.

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Section: Resultssupporting

confidence: 92%

TEM1 up-regulates MMP-2 and promotes ECM remodeling for facilitating invasion and migration of uterine sarcoma

Sun

Shen

et al. 2023

Discov Onc

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“…The assays were performed according to the protocol described by Mazurkiewicz et al [ 24 ]. Poly-L-lysine-coated coverslips (Corning) were washed with PBS and then fixed with 0.5% glutaraldehyde for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Melanoma cells with diverse invasive potential differentially induce the activation of normal human fibroblasts

et al. 2022

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Background The tumor microenvironment consists of stromal cells, extracellular matrix, and physicochemical properties (e.g., oxygenation, acidification). An important element of the tumor niche are cancer-associated fibroblasts (CAFs). They may constitute up to 80% of the tumor mass and share some features with myofibroblasts involved in the process of wound healing. CAFs can facilitate cancer progression. However, their interaction with melanoma cells is still poorly understood. Methods We obtained CAFs using conditioned media derived from primary and metastatic melanoma cells, and via co-culture with melanoma cells on Transwell inserts. Using 2D and 3D wound healing assays and Transwell invasion method we evaluated CAFs’ motile activities, while coverslips with FITC-labeled gelatin, gelatin zymography, and fluorescence-based activity assay were employed to determine the proteolytic activity of the examined cells. Western Blotting method was used for the identification of CAFs’ markers as well as estimation of the mediators of MMPs’ (matrix metalloproteinases) expression levels. Lastly, CAFs’ secretome was evaluated with cytokine and angiogenesis proteomic arrays, and lactate chemiluminescence-based assay. Results Acquired FAP-α/IL6-positive CAFs exhibited elevated motility expressed as increased migration and invasion ratio, as well as higher proteolytic activity (area of digestion, MMP2, MMP14). Furthermore, fibroblasts activated by melanoma cells showed upregulation of the MMPs’ expression mediators’ levels (pERK, p-p38, CD44, RUNX), enhanced secretion of lactate, several cytokines (IL8, IL6, CXCL1, CCL2, ICAM1), and proteins related to angiogenesis (GM-CSF, DPPIV, VEGFA, PIGF). Conclusions Observed changes in CAFs’ biology were mainly driven by highly aggressive melanoma cells (A375, WM9, Hs294T) compared to the less aggressive WM1341D cells and could promote melanoma invasion, as well as impact inflammation, angiogenesis, and acidification of the tumor niche. Interestingly, different approaches to CAFs acquisition seem to complement each other showing interactions between studied cells.

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“…Fluorescent-gelatin degradation assays were performed according to the protocol described by Mazurkiewicz et al [ 29 ]. Poly-L-lysine-coated coverslips (Corning) were washed with PBS and fixed with 0.5% glutaraldehyde for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Melanoma stimulates the proteolytic activity of HaCaT keratinocytes

et al. 2022

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Background Keratinocytes constitute a major part of the melanoma microenvironment, considering their protective role towards melanocytes in physiological conditions. However, their interactions with tumor cells following melanomagenesis are still unclear. Methods We used two in vitro models (melanoma-conditioned media and indirect co-culture of keratinocytes with melanoma cells on Transwell inserts) to activate immortalized keratinocytes towards cancer-associated ones. Western Blotting and qPCR were used to evaluate keratinocyte markers and mediators of cell invasiveness on protein and mRNA expression level respectively. The levels and activity of proteases and cytokines were analysed using gelatin-FITC staining, gelatin zymography, chemiluminescent enzymatic test, as well as protein arrays. Finally, to further study the functional changes influenced by melanoma we assessed the rate of proliferation of keratinocytes and their invasive abilities by employing wound healing assay and the Transwell filter invasion method. Results HaCaT keratinocytes activated through incubation with melanoma-conditioned medium or indirect co-culture exhibit properties of less differentiated cells (downregulation of cytokeratin 10), which also prefer to form connections with cancer cells rather than adjacent keratinocytes (decreased level of E-cadherin). While they express only a small number of cytokines, the variety of secreted proteases is quite prominent especially considering that several of them were never reported as a part of secretome of activated keratinocytes’ (e.g., matrix metalloproteinase 3 (MMP3), ADAM metallopeptidase with thrombospondin type 1 motif 1). Activated keratinocytes also seem to exhibit a high level of proteolytic activity mediated by MMP9 and MMP14, reduced expression of TIMPs (tissue inhibitor of metalloproteinases), upregulation of ERK activity and increased levels of MMP expression regulators-RUNX2 and galectin 3. Moreover, cancer-associated keratinocytes show slightly elevated migratory and invasive abilities, however only following co-culture with melanoma cells on Transwell inserts. Conclusions Our study offers a more in-depth view of keratinocytes residing in the melanoma niche, drawing attention to their unique secretome and mediators of invasive abilities, factors which could be used by cancer cells to support their invasion of surrounding tissues.

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A Fluorescent Gelatin Degradation Assay to Study Melanoma Breakdown of Extracellular Matrix

Cited by 6 publications

References 21 publications

TEM1 up-regulates MMP-2 and promotes ECM remodeling for facilitating invasion and migration of uterine sarcoma

TEM1 up-regulates MMP-2 and promotes ECM remodeling for facilitating invasion and migration of uterine sarcoma

Melanoma cells with diverse invasive potential differentially induce the activation of normal human fibroblasts

Melanoma stimulates the proteolytic activity of HaCaT keratinocytes

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