Simple and Cost-Effective Assay for Isolating Invasive Living Cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.

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“…To investigate the role of TGF-β1-induced proMMP-9 activation on cell invasion, we employed a novel invasion assay [ 16 , 17 ] ( Fig. 4 A).…”

Section: Resultsmentioning

confidence: 99%

“…The iCT assay was performed as described previously [ 16 , 17 ]. Cell strainers with a nylon mesh of 40-μm 2 square pores (Funakoshi, FN-HT-AMS-14002, Tokyo, Japan) were placed in an inverted orientation in a culture dish (100-mm diameter).…”

Section: Methodsmentioning

confidence: 99%

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Yamahana

Terashima

Takatsuka

et al. 2021

Biochemistry and Biophysics Reports

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“…As such, this method enables the quantification of the biofragmentation step by monitoring PLA weight loss. This new method for the quantification of plastic biodegradation is a new application for cell strainers, which have already found applications in various fields ( Qiu et al, 2018 ; Takino et al, 2018 ; Wang et al, 2019 ). To the authors’ knowledge, this is the first study reporting on the combined use of cell strainers and sonication to analyze the biodegradation of plastics.…”

Section: Resultsmentioning

confidence: 99%

Novel methods to monitor the biodegradation of polylactic acid (PLA) by Amycolatopsis orientalis and Amycolatopsis thailandensis

Yasin,

Pancho,

Yasin

et al. 2024

Front. Bioeng. Biotechnol.

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Plastics are essential in modern life, but their conventional production is problematic due to environmental pollution and waste management issues. Polylactic acid (PLA) is a widely used bioplastic that is bio-based and biodegradable, making it a key player in the bioeconomy. PLA has been proven to be degradable in various settings, including aqueous, soil, and compost environments. However, monitoring and optimizing PLA biodegradation remains challenging. This study proposes methods to improve the quantification of PLA biodegradation by Amycolatopsis spp. Ultrasound treatments (10 s) significantly improved the enumeration of viable Amycolatopsis cells by breaking the pellets into quantifiable individual cells. A separation technique combining ultrasound (120 s) and 40 μm cell strainers effectively isolated PLA particles from biomass to quantify PLA weight loss. This enabled the monitoring of PLA biofragmentation. Finally, CO2 production was measured according to ISO 14852 to quantify mineralization. Integrating these methods provides an improved quantification for PLA biodegradation along its different stages. In a case study, this led to the construction of a carbon balance where 85.1% of initial carbon content was successfully tracked. The developed techniques for monitoring of PLA biodegradation are essential to design future waste management strategies for biodegradable plastics.

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“…To address this issue, we developed a novel 3D model to quantify cell invasion: the invading cell-trapping (iCT) assay (Takino, Sato, & Seiki, 2018). By using an innovatively designed chamber, this novel 3D invasion assay permits quantitative measurement of cell invasion from a primary gel into a secondary gel.…”

Section: Introductionmentioning

confidence: 99%

Isolation of Highly Migratory and Invasive Cells in Three‐Dimensional Gels

2020

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We developed a modified invasion assay in three‐dimensional (3D) gels that permits isolation of invading cells as living cells, termed an invading cell–trapping (iCT) assay. A small cell strainer consisting of nylon mesh with pores of 40‐µm square is used in this assay. A layer of gel composed of extracellular‐matrix components is formed on each side of the nylon mesh, which permits cell migration or invasion from one gel layer to the other. At the end of the assay, the two gel layers are removed from the apparatus and easily separated from each other. Invading cells from the primary gel are trapped in the secondary gel, which maintains the morphology and other properties of the invasive cells in a 3D matrix. The cells that have invaded are observed and counted with a standard light microscope without cell staining. There is no need for a specialized microscope, imaging analysis software, or advanced cell‐biological technical knowledge in this assay. This assay can also reduce measurement of nonspecific cell invasion by monitoring the upward invasion of cells. The viability of both invading and non‐invading cells trapped in the gels can be assessed by typical colorimetric assays, if desired. This assessment characterizes the total number of cells (invading and non‐invading cells) and the ratio of invading cells to total cells. By repeating the iCT assay, further enrichment of invasive and noninvasive cells can be attained. Thus, this assay improves comparative analyses between invasive and noninvasive cells. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring upward cell invasion into collagen gel Basic Protocol 2: Measuring cell invasion from Matrigel into collagen gel Basic Protocol 3: Isolation and enrichment of highly invasive cells

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Simple and Cost-Effective Assay for Isolating Invasive Living Cells

Cited by 5 publications

References 20 publications

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Novel methods to monitor the biodegradation of polylactic acid (PLA) by Amycolatopsis orientalis and Amycolatopsis thailandensis

Isolation of Highly Migratory and Invasive Cells in Three‐Dimensional Gels

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