Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells
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26
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Fuse (electrical)Electric power distributionParticle physicsVideoMarch (territory)Plain bearingRestkernBiomedical engineeringComputer animation
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Musical developmentProgressive lensProzessleittechnikTARGET2MechanicTrainLight
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Substrate (printing)Ship classCartridge (firearms)
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ProzessleittechnikGroup delay and phase delayBlood vesselTissue paperWeightAcoustic membraneForce
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Scale (map)GlassPulse-amplitude modulationMembrane potentialAngle of attackParticleTiefdruckgebietTypesettingMechanicMembrane potentialComputer animation
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KoerzitivfeldstärkeGelVideoGelKoerzitivfeldstärkeComputer animation
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ParticleKoerzitivfeldstärkeGelParticleAccess networkComputer animation
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GelAerodynamicsHandle (grip)Plain bearingModel buildingVideoField-effect transistorHot workingParticleSizingPlane (tool)Model buildingParticle physicsPlain bearing
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Membrane potentialRestkernScale (map)RestkernGelBahnelementForceCountersinkScale (map)Speckle imagingSchwache LokalisationAtomic nucleusFocal lengthHot workingRutschungComputer animation
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Hull (watercraft)RestkernMembrane potentialAtomic nucleusHull (watercraft)Effects unitRestkernForceHyperbelnavigationStress (mechanics)Band gapRutschungDiagram
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Atomic nucleusHull (watercraft)Hull (watercraft)GelRestkernHot workingDiagram
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ProzessleittechnikForceSpeckle imagingLecture/Conference
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Biomedical engineeringComputer animation
Transcript: English(auto-generated)
00:10
Cancer development and progression is a difficult problem leading to worldwide mortality. While the genetics and the biochemistry of the problem have been studied intensely,
00:21
the biophysics also has important meaning. Specifically, the mechanical interactions of cancer cells with their environment can reveal how these cells perform processes related to invasion and allow us to understand how they successfully invade new regions and then proliferate. This could reveal targets for treatment and allow us to manage and diagnose this problem more easily.
00:46
In this work, we'll show an automated approach to quantitatively analyze focal images, which allows us to identify differences between invasive metastatic cancer cells and benign cells and be able to show how they mechanically interact differently with their substrates.
01:06
During metastasis, a single cell that is shed from the primary tumor or a group of cells will push their way through tissue containing other cells and membrane, swim through the blood vessel or the lymph nodes, push out, successfully adhere,
01:24
and then metastasize, generating a secondary tumor at a faraway site. This process relies heavily on changes in both cell stiffness as well as their ability to apply force, so interactions with their environment are critical.
01:41
The way that we will be testing the mechanical interaction of cancer cells with their environment is through an in vitro experimental system, including an impenetrable gel composed of polyacrylamide, which has 200-nanometer fluorescent particles embedded at its top,
02:02
and collagen type 1 as a monolayer on the top layer to facilitate cell adherence. We will be comparing three different cell types, a high metastatic potential cell, low metastatic potential breast cancer cell, and benign cells. When placing all three cell lines on these gels, the cells morphologically look similar,
02:24
as cells on soft gels typically remain rounded and do not spread out. However, when looking at the location of the particles underneath the cells, we have observed an interesting phenomenon. In the benign cells, we may observe the particles being moved laterally on the XY surface of the gel.
02:48
However, underneath the low and high metastatic potential cells, we observe a hole. Since these are impenetrable gels, they are non-degradable and the pores are too small, cells do not have access to the particles.
03:03
What actually happens here is that the cells are indenting the gels, moving the particles to a lower focal plane. As we had previously observed in a work also published in New Journal of Physics, the cells will grip onto the gel, apply force, and pull the gel inwards and upwards,
03:23
pushing the entire cell body downwards into the gel. Using the Hertz model and with the cell morphology sizes and the gel stiffness, we were able to calculate the forces applied by each single cell as a function of time.
03:41
In the current work, we are interested in how these cells apply force to successfully indent the gels, specifically looking at changes in cell morphology, localization of the nucleus and the cytoskeleton. To do this, we have utilized a custom-written software based in MATLAB 2012
04:01
that allows us to take confocal image slices and perform 3D rendering and then automatically measure scales of the cells and the different stained elements in the cell. In the current slide, what you can see is the gel surface and the cell nuclei,
04:20
without showing the cell membrane, just for clarity. The difference is already observed between benign cells, where the gels are only slightly indented, if at all, where the nucleus is rounded, and high metastatic potential cells, where the nuclei are in a different shape and within the indentation dimple.
04:40
In the manuscript, we show reorganization of the cytoskeleton and the nucleus likely to facilitate force application. Our main interest was the indentation depth and how the nucleus participates in this process. In the current slide, we show that with our automated image processing approach, we are able to identify indentation depths that are much larger in the metastatic cancer cells than in the benign cells.
05:07
We speculate that any indentation depths observed in the benign cells likely occurs as a side effect of their applying lateral traction forces and the gel collapsing underneath them. So we define the maximal indentation depth identified in the benign cells,
05:25
which is about 2.7 microns, as an unintentional indentation depth, focusing on indentation depths that are larger than that, that are observed in the cancer cells. Looking at where the nucleus is relative to the indentation depth,
05:41
we observe that regardless of the depth in the benign cells, the nucleus is always above the gel. However, in the metastatic cancer cells, as the cells indent the gels deeper than this unintentional indentation depth observed in the benign cells, suddenly the nuclei appear to be participating in some sort of way or actually being pulled into the gel indentation.
06:06
In this work, we have shown that as invasive metastatic cancer cells apply force to their underlying gel, a coordinated process occurs involving the cytoskeleton and the nucleus. The automated image processing approach that we have introduced and shown here
06:21
can also be used to analyze different images with different processes occurring in cells.