Visualizing Cancer Cell Chemotaxis and Invasion in 2D and 3D

Susanto, Olivia; Muinonen-Martin, Andrew; Nobis, Max; Insall, Robert H.

doi:10.1007/978-1-4939-3480-5_17

Cited by 6 publications

(7 citation statements)

References 11 publications

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“… Spread assay to assess LPA breakdown and chemotaxis. (A) (i) Side view of an Insall chemotaxis chamber showing inner and outer wells and positioning of the coverslip with cells adhered to it (adapted from Susanto et al, 2016 ); (ii) bird's eye view of an Insall chamber, indicating the position of the viewing bridges (in yellow). (B) A uniform serum environment produces differential directional movement of the cells on the left (left cluster; i–iii) and right (right cluster; iv–vi) of the bridge, as quantified by mean (±s.e.m.)…”

Section: Resultsmentioning

confidence: 99%

“…Cells were plated onto fibronectin-coated glass coverslips at a density of 7×10 4 cells/ml and then serum-starved overnight in serum-free RPMI medium prior to use as previously described ( Muinonen-Martin et al, 2013 ; Susanto et al, 2016 ). Serum-free (SF) medium consisted of RPMI with 2 mM l -glutamine, penicillin/streptomycin (Invitrogen) and 0.5% BSA (Sigma), while serum-containing medium contained an additional 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

“…The CIA was carried out as previously described ( Yu and Machesky, 2012 ; Susanto et al, 2016 ). Briefly, an Ibidi culture insert was adhered to the centre of a 35 mm low μ-dish (Ibidi), then 1×10 6 WM1158 cells were plated in the dish around the insert.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Susanto

Koh

Morrice

et al. 2017

Journal of Cell Science

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Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 – but not of its homologues LPP1 or LPP2 – diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering.This article has an associated First Person interview with the first author of the paper.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Susanto

Koh

Morrice

et al. 2017

Journal of Cell Science

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“…Most laboratory assays for chemotaxis [for example, Zigmond/Dunn/Insall chambers (Zigmond and Hirsch, 1973;Zicha et al, 1991;Muinonen-Martin et al, 2010), transwell assays (Zigmond and Hirsch, 1973) and single-track under-agarose assays as shown in Figure 2B] are inherently one-dimensional; a group of cells follow a path of essentially constant width. In 2D assays such as circular invasion (Yu and Machesky, 2012;Susanto et al, 2016) or one-spot assays (Figure 3A), the migration front of the chemotactic cells constantly increases in circumference, so low cell density will inevitably be a problem after cells have migrated a significant distance. Three-dimensional assays are even more profoundly affected because the front expands more rapidly.…”

Section: D and 3d Chemotaxis Add A Level Of Complexitymentioning

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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“…Cells were plated on fibronectin-coated glass coverslips at a density of 7x10 4 cells/ml then serum starved overnight in serum-free RPMI medium prior to use as previously described [18,33]. Serum-free (SF) medium comprised RPMI with 2mM L-glutamine, penicillin/streptomycin (Invitrogen) and 0.5% BSA (Sigma), while serumcontaining medium contained an additional 10% FBS.…”

Section: Insall Chamber Chemotaxis Assaymentioning

confidence: 99%

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

Susanto

Koh

Morrice

et al. 2017

Preprint

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Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 -but not of its homologues LPP1 or LPP2 -diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering.This article has an associated First Person interview with the first author of the paper.

show abstract

Visualizing Cancer Cell Chemotaxis and Invasion in 2D and 3D

Cited by 6 publications

References 11 publications

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

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

Self-Generated Gradients Yield Exceptionally Robust Steering Cues

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

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