LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

Susanto, Olivia; Koh, Yvette W. H.; Morrice, Nick; Tumanov, Sergey; Thomason, Peter A.; Nielson, Matthew; Tweedy, Luke; Muinonen-Martin, Andrew; Kamphorst, Jurre J.; Mackay, Gillian; Insall, Robert H.

doi:10.1242/jcs.207514

Cited by 41 publications

(36 citation statements)

References 39 publications

(50 reference statements)

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“…In the current work we only consider gradients established by cell breaking down broadly distributed attractants. Cell typically use two different methods to drop the local concentrations -they can endocytose receptors when they are bound to ligands (Donà et al, 2013), thereby removing the ligand for proteolysis or chemical breakdown, or they can use cell-surface enzymes like MMPs (McQuibban et al, 2002), lipid phosphatases (Susanto et al, 2017) and phosphodiesterases (Garcia et al, 2009). The endocytosis pathway can be reinforced by specific "scavenger" receptors, which remove ligand without necessarily transducing a signal, as occurs when the zebrafish lateral line primodium migrates (Donà et al, 2013;Venkiteswaran et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Self-Generated Gradients Yield Exceptionally Robust Steering Cues

Tweedy

Insall

2020

Front. Cell Dev. Biol.

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Chemotaxis is a widespread mechanism that allows migrating cells to steer to where they are needed. Attractant gradients may be imposed by external sources, or selfgenerated, when cells create their own steep local gradients by breaking down a prevalent, broadly distributed attractant. Here we show that chemotaxis works far more robustly toward self-generated gradients. Cells can only respond efficiently to a restricted range of attractant concentrations; if attractants are too dilute, their gradients are too shallow for cells to sense, but if they are too high, all receptors become saturated and cells cannot perceive spatial differences. Self-generated gradients are robust because cells maintain the attractant at optimal concentrations. A wave can recruit varying numbers of steered cells, and cells can take time to break down attractant before starting to migrate. Self-generated gradients can therefore operate over a greater range of attractant concentrations, larger distances, and longer times than imposed gradients. The robustness is further enhanced at low cell numbers if attractants also act as mitogens, and at high attractant concentrations if the enzymes that break down attractants are themselves induced by constant attractant levels.

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Section: Introductionmentioning

confidence: 99%

Self-Generated Gradients Yield Exceptionally Robust Steering Cues

Tweedy

Insall

2020

Front. Cell Dev. Biol.

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“…The random motility of the amoebae was observed for 30 min, taking frames every 30 sec using a Zeiss 700 series microscope equipped with a 10x air objective. Automatic cell tracking was performed as described previously (Susanto et al, 2017).…”

Section: Methodsmentioning

confidence: 99%

Akt kinases are required for efficient feeding by macropinocytosis inDictyostelium

Williams

Peak‐Chew

Paschke

et al. 2018

Preprint

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Macropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.Summary statementDictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.

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Section: Thus Our Indices Reveal That Tumor Cell Invasion Is Facilitmentioning

confidence: 99%

“…First, the most common analysis is to measure the cell-covered area, mainly a relative area to a control (i.e. day 0 or untreated sample) 4,18,19,22,[24][25][26] . Second, measurements of the maximum length, or the distance from the edge of the initial spheroid to the outermost edge of the protruding cells, are often performed 21,27,28 .…”

Section: Comparison Of Invasion Indices With Parameters Of Conventionmentioning

confidence: 99%

“…In particular, the edges of the invasive areas in cellular invasion assays are often not defined unambiguously. While recent quantitative analyses have employed diverse digital filters or computational parameters such as texture 4,17 , in many studies of cellular invasion, rather simple parameters such as the maximum distance that cells migrate or the area covered by the migrating cells are still preferred [18][19][20][21][22] .Here, we provide novel quantitative indices for the invasiveness of cancer cells grown in spheroid culture. The indices are calculated from an image-based spheroid invasion assay that consists of (1) obtaining brightfield images with a standard inverted microscope, (2) setting objective threshold values for image analysis, and (3) segmenting a cell-covered area into a few discrete areas depending on cell density.…”

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confidence: 99%

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Novel invasion indices quantify the feed-forward facilitation of tumor invasion by macrophages

Lim

Kang

Lee

2020

Sci Rep

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Quantitative and reliable measurement of cellular invasion is important to understand a range of biological processes such as cancer metastasis and angiogenesis. Spheroid invasion assays are an attractive in vitro platform because they effectively mimic the tumor cell invasion of solid tissues. Here, we developed an image analysis-based method to quantify the invasiveness of HT1080 human fibrosarcoma tumor cell spheroids. We segmented a cell-covered area into three subareas using objectively set threshold pixel intensities and calculated invasion indices using these subareas. comparison with conventional parameters for spheroid invasion assays, such as area, length, and detached cells, showed that our indices present the invasion event at an early time and without being convoluted by proliferation. As an application, we then examined paracrine interactions between LLC1 mouse lung carcinoma cells and Raw264.7 mouse macrophage cells with our developed analysis method. We found that the invasion of tumor spheroids was increased by a macrophage-conditioned medium, concomitantly with a decrease in tumor cell proliferation. importantly, invasion was further enhanced by a conditioned medium from activated macrophages by co-culture with tumor cells. thus, our indices reveal that tumor cell invasion is facilitated in a feed-forward manner by communication between tumor cells and macrophages in the tumor microenvironment.Cellular migration and invasion are essential in many biological processes such as cancer metastasis 1-3 and angiogenesis by endothelial cells 4 . Several assays have been developed to measure cellular invasion 3 ; basically, the invasiveness of cells is measured by the distance or the path that cells travel in different setups such as Boyden chambers or wound healing assays. However, the cells are generally grown in two-dimensional (2D) in vitro space, which does not fully recapitulate the behavior of cells grown in three-dimensional (3D) in vivo tissue. Recently, spheroid-based 3D cultures have been widely employed in many applications, particularly drug discovery, as an in vivo-mimicking cellular platform 5-9 . In spheroid culture, cellular interactions among cells or with the extracellular matrix are better preserved 10,11 , and in addition, the physiological gradients inside of the tumor spheroid, such as oxygen, pH, and nutrients, create an environment that better mimics in vivo.To date, most analyses of the invasiveness of cells grown in spheroids have been qualitative and descriptive rather than quantitative [12][13][14] . Raw data of cellular invasion usually takes the form of micrographs that are taken from bright field microscopy, with subsequent quantitative analysis of the micrographs relying on computational image processing accompanied by an impartial determination of the key parameters. Although there are good examples of image analysis of bright field micrographs, such as yeast growth 15 and stem cell proliferation 16 analyses, accurate quantitation based on bright field micrographs is cha...

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LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

Cited by 41 publications

References 39 publications

Self-Generated Gradients Yield Exceptionally Robust Steering Cues

Self-Generated Gradients Yield Exceptionally Robust Steering Cues

Akt kinases are required for efficient feeding by macropinocytosis inDictyostelium

Novel invasion indices quantify the feed-forward facilitation of tumor invasion by macrophages

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