Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Cvetković, Donna; Goertzen, Cameron; Bhattacharya, Moshmi

doi:10.3791/51341-v

Cited by 4 publications

(4 citation statements)

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“…MDA‐MB‐231 cells were embedded in 50% Matrigel matrix (BD Biosciences) and processed for fluorescent staining to assess changes in invasive morphological features as described by Cvetković et al (2014). In summary, 35 mm Glass‐bottom cell culture dishes (MatTek) were coated with 50% Matrigel (BD Bioscience) in serum‐free medium.…”

Section: Methodsmentioning

confidence: 99%

“…Cell colony morphology was monitored for five days using bright‐field microscopy (Leica DM16000 B microscope with Leica DFC425 camera) at 10X magnification, and representative 50 μm z‐stack images (2 μm interval) taken at random locations across the matrix were captured using Leica MMAF software (Metamorph®). Fluorescent staining procedure was done as described in Cvetković et al (2014) using Alexa Fluor 633 phalloidin (1:100) and DAPI (1 μg/mL) staining. Samples were imaged using a Nikon A1R+ confocal microscope and NIS Elements software (Nikon), capturing Z‐stacks of approximately 100 μm.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels

Cepeda

Evered

Pelling

et al. 2017

J. Cell Commun. Signal.

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Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14) is a unique protease that cleaves extracellular proteins, activates proMMPs, and initiates intracellular signalling. MCF-7 cells are non-invasive and deficient in MT1-MMP, MMP-2, and MMP-9 expression. We created an MCF-7 cell line (C2) that stably produces active MT1-MMP and demonstrated increased ERK1/2 phosphorylation. MAPK inhibition in this cell line showed an inverse relationship in MMP-2 and MMP-9 transcripts where levels of these genes increased and decreased, respectively. Using invasive MDA-MB 231 cells that endogenously produce MT1-MMP and have naturally high pERK levels, we demonstrated the identical inverse relationship between MMP-2 and -9 transcript and protein levels, suggesting that this novel relationship is conserved amongst MT1-MMP positive breast cancer cells. To further analyze the relationship between MMP-2 and -9 levels, we chemically inhibited activation and catalytic activity of MT1-MMP using a furin and MMP inhibitor, respectively, to show that interference with the functions of MT1-MMP induced changes in MMP-2 and 9 transcript levels that were always inverse of each other, and likely mediated by differential transcriptional activity of the NF-κB transcription factor. Furthermore, we analyzed the functional consequences of these expression changes to show MMP, and in particular ERK, inhibition decreased migration and invasion using 2D culture, and inhibits the formation of an invasive phenotype in Matrigel 3D culture. This study demonstrated a novel inverse transcriptional relationship between MMP-2 and -9 levels and MT1-MMP activity that have functional consequences, and also showed that increases in the levels of MMPs does not necessarily correlate with an invasive phenotype.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels

Cepeda

Evered

Pelling

et al. 2017

J. Cell Commun. Signal.

View full text Add to dashboard Cite

show abstract

“…Cells were embedded in 50% Matrigel matrix (BD Biosciences) and examined for changes in invasive morphological features as described by Cvetković et al (2014). 35 mm glass‐bottom cell culture dishes (MatTek) were coated with 50% Matrigel in serum‐free medium.…”

Section: Methodsmentioning

confidence: 99%

Stable expression of α1-antitrypsin Portland in MDA-MB-231 cells increased MT1-MMP and MMP-9 levels, but reduced tumour progression.

Willson

Muir

Evered

et al. 2017

J. Cell Commun. Signal.

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The membrane bound matrix metalloproteinase MT1-MMP plays roles in modulating cell movement, independent of its abilities to remodel the extracellular matrix. Unlike many MMPs, MT1-MMP is activated in the Golgi prior to secretion by a pro-protein convertase, primarily furin. Regulation of the activation of pro-MT1-MMP has been methodically investigated, as altering the level of the active protein has broad implications in both activating other pro-MMPs, including pro-MMP-2, and many subsequent remodelling events. Our previous work in MCF-7 cells has demonstrated that modest, and not extremely high, levels of active MT1-MMP manifests into altered cell morphology and movement. At this low but optimal amount of MT1-MMP protein, changes to MT1-MMP levels are always mirrored by MMP-9 and pERK levels, and always opposite to MMP-2 levels. In this study, stable expression of the furin inhibitor α1-antitrypsin Portland (α1-PDX) in MDA-MB-231 cells increased overall MT1-MMP levels, but cells maintained a 21% proportion of pro-MT1-MMP. The increase in MT1-MMP was mirrored by increases in MMP-9 and pERK, but a decrease in MMP-2. These changes were associated with increased NF-κB transcription. In vitro analysis showed that α1-PDX decreased cell protrusions and migration, and this manifested as decreased tumourigenesis when examined in vivo using a chick CAM assay.

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“…For instance, an antimicrobial peptide may promote the cell growth of malignant cells (Table 1). Taking BST2, GAPDH, S100A8, S100A9 and HMGB1 as examples, in spite of their advantageous role in different infections (Supplementary Data 1), they contribute to the cancer cell proliferation, invasiveness, poor survival, and disease severity in different cancers [33-36, 43, 48, 49]. In addition, S100A7 has a dual behavior in different malignancies.…”

Section: Discussionmentioning

confidence: 99%

Drug repositioning for immunotherapy in breast cancer using single-cell and spatial transcriptomics analysis

Mohammadi

Jin

Zhang

et al. 2022

Preprint

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Immunomodulatory peptides are capable of stimulating or suppressing the immune system. Hence, deregulation of them can be considered as an immunotherapy approach. These peptides may have dual behavior in response to different infections. For instance, an antimicrobial peptide may act as an anticancer, tumor marker or even cause cancer progression. In this study, we used single-cell RNA sequencing analysis to investigate the deregulation of immunomodulatory peptides in malignant versus normal human breast epithelial cells. We validated the obtained results in chromatin accessibility level. Furthermore, we used a drug repositioning approach to change the expression of these peptides based on their role in cancer biology. As a result, ten immunomodulatory peptides were upregulated in breast cancer versus normal. Chromatin was more accessible for these peptides in cancer cell lines versus normal. Among these ten peptides, five of them were tumor drivers (i.e., BST2, GAPDH, S100A8, S100A9 and HMGB1), three of them were anticancer (i.e., H2AFJ, SCGB2A1 and HMGN2), S100A7 had dual behavior in different cancers and ZG16B was a tumor marker. Using the LINCS L1000 database, we proposed a list of drugs that can deregulate the candidate peptides according to their role in the progression of malignancy. In conclusion, immunomodulatory peptides can be considered as drug targets based on their role in cancer biology.

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Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Cited by 4 publications

References 0 publications

Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels

Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels

Stable expression of α1-antitrypsin Portland in MDA-MB-231 cells increased MT1-MMP and MMP-9 levels, but reduced tumour progression.

Drug repositioning for immunotherapy in breast cancer using single-cell and spatial transcriptomics analysis

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