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Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

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Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells
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Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.
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Prozessleittechnik Speckle imaging Gel Biomedical engineering Force
insulin and regression using I mean to I will tell you a lot of genetics and I use it all and and intensely in the light of this also has important the specifically the mechanical interaction itself has resulted in violence in real time these cells or processes belatedly Asian and allows us to understand how a successfully age you read this and then a for it this is a real targets for training and allows to manage and notes this problem in this
world shown on the 1st class you have arise from full reduce which allows us to get by differences in cases that and so then I'm cells and be able to show that we can apps differently with their substrates
during metastasis a single cell that is shed from the primary tumor or 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 and here and there and metastasis eyes generating a secondary tumor at a far away site this process relies heavily on changes in both cell stiffness as well as their ability to apply force so interactions with the environment are critical the weight that
we will be testing their interaction the mechanical interaction of cancer cells with their environment this through an in vitro experimental system including in impenetrable gel composed of polyacrylamide which has 200 enemy a fluorescent particles embedded at its top and collagen type 1 as a monolayer on the top layer to facilitate cell adherence we will be comparing 3 different cell types of a high metastatic potential cell low metastatic potential breast cancer cell and benign
cells when placing all 3 cell lines on these jails the cells morphologically look similar as cells on soft-shells typically remain rounded in and do not spread out however when looking at
this is the location of the particles underneath the cells we have observed an interesting phenomena in the 9 cells we may observe that the particles being moved laterally on the x-y surface of the gel however underneath the low and high metastatic potential cells we observe a whole since these are impenetrable jails they are non-degradable and the porter too small cells do not have access to the particles what actually happens here
is that the cells are indenting the jails of moving the particles to a lower focal plane as we had previously observed in a work also published a new Journal of Physics the cells will grab onto the gel apply for and pull the gel in words in upwards pushing the entire cell body downwards into the gel using the Hertz model and with the cell morphology sizes and the jails that this we were able to calculate the forces applied by each single cell as a function of time in the
current work we're interested in how the cells apply force to successfully and then the jails specifically looking at changes in cell morphology localization of the nucleus and the cytoskeleton to do this we have utilized a custom-written software maced in MATLAB 2012 that allows us to take could focal image sizes and perform 3-D rendering and then automatically measure the scales of the cells and the different stained elements in the cell and the current slide we can see is the gel surface and the cell nuclei without showing the cell membrane just for clarity the difference is already observed between the 9 cells where the jails are only slightly indented if and all were the nucleus is rounded and high metastatic potential cells were the nuclei are or in a different shape and within the indentation dimple in
the manuscript we show reorganization of the cytoskeleton and the nuclear slightly to facilitate force application our main interest was the indentation depth and how the nucleus participates in this process the current slide we show that with the automated image processing approach we are able to identify indentation gaps there much larger in the metastatic cancer cells then the B 9 cells 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 to we defined the maximal indentation depth identified in the the 9 cells which is about 2 comma decimal 7 microns as an unintentional indentation depth focusing on in addition debts that are larger than that the observed in the cancer cells looking at where the nucleus
is relative to the indentation that we observe that regardless of the depths in the benign cell the nucleus is always about the gel however in the metastatic cancer cells as the cells in dense the jails deeper than this so unintentional indentation depths observed in the benign cells suddenly the nuclei appear to be participating in some sort of way are actually being pulled into the gel indentation in this work we have shown that it is
invasive metastatic breast cancer cells apply a force to the underlying gel according to process occurs the cytoskeleton includes nomination image processing a person you know introducing shown here can also be used to analyze different images with different processes occurring cells