Cancer cell mechanics

Haruka Yoshie

NMuMG-NT cells treated with TGF-β

Cells migrate in our body from when we are first being formed to when we are recovering from injuries as well as when we are sick. For example, our primitive organs are formed by the movement of specific cells during the embryonic development. During immune response, immune cells move in search of foreign organisms to fight against infections. To close the open wounded site, epithelial cells migrate across the injured site while establishing new cell-cell contacts [1]. Although cell motility enables us to develop, cure and heal, it can also have negative implications as is the case in cancer metastasis. During cancer metastasis, cancer cells travel from their primary site to secondary sites, spreading the disease in our body. This metastatic behaviour of cancer accounts for 90% of cancer-related deaths. Previously, some studies have shown that cancer cells applied larger traction stresses than non-metastatic cells [2] while others have observed an inverse relationship between the metastatic potential and traction stresses [3]. Additionally, other studies have shown cancer cells are softer than benign cells [4]. These results suggest that there is a link between the cancer disease and cellular mechanics. In our studies, we would like to better understand the physical and mechanical changes in the cancer disease. 

One of the physical mechanisms we are particularly interested in is Epithelial to Mesenchymal Transition (EMT). An induction of EMT program has been proposed as a crucial mechanism for malignant epithelial cancer cells causing tumor cells to become more motile and invasive allowing them to spread in our body [5]. During the EMT process, epithelial cells lose their cell polarity and cell-cell adhesions while gaining mesenchymal properties including enhanced migratory and invasive behaviour. However, physical changes during EMT are still not well understood. We are interested in investigating how cell mechanics change during EMT process. Our research specifically focuses on how cellular forces and work change during EMT. In our studies, we examine how mechanical properties evolve during this transition utilizing soft silicone substrates. We are also interested in studying how cells change mechanical properties during EMT. To study the mechanical changes of the cells, we will utilize the magnetic twisting cytometry and the passive microrheology. Additionally, we investigate how cytoskeletal structures change as cells undergo EMT by focusing on the structural changes of actin stress fibers and it affects the changes in physical properties of the cells.

Overview of multi-well plate fabrication. (A) A custom-cut glass slide is the starting point. (B) The glass slide is coated with a thick (~100 μm layer of PDMS). (C) A layer of fiduciary beads (shown in green) are then spin-coated in a ~1 μm thick layer on top of the previous layer. (D) The multi-well divider is carefully placed on top of the fiduciary bead layer. (E) The complete multi-well plate is assembled and ready for use or storage. [6]

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications, however, require direct quantification of these forces. We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems. We use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-β, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-β exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics. [6]

1.   Lodish, H., Molecular Cell Biology. W. H. Freeman: 2008.

2.   Kraning-Rush, C. M.; Califano, J. P.; Reinhart-King, C. A., Cellular traction stresses increase with increasing 

      metastatic potential. PloS one 2012, 7 (2).

3.   Indra, I.; Undyala, V.; Kandow, C.; Thirumurthi, U.; Dembo, M.; Beningo, K. A., An in vitro correlation of 

      mechanical forces and metastatic capacity. Physical biology 2011, 8 (1).

4.   Cross, S. E.; Jin, Y. S.; Rao, J.; Gimzewski, J. K., Nanomechanical analysis of cells from cancer patients. 

      Nature nanotechnology 2007, 2 (12), 780-3; (b) Guo, M.; Ehrlicher, A. J.; Jensen, M. H.; Renz, M.; Moore, J. R.; 

      Goldman, R. D.; Lippincott-Schwartz, J.; Mackintosh, F. C.; Weitz, D. A., Probing the stochastic, motor-driven 

      properties of the cytoplasm using force spectrum microscopy. Cell 2014, 158 (4), 822-32.

5.   Yilmaz, M.; Christofori, G., EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev Cancer 

      and Metastasis Reviews 2009, 28 (1-2), 15-33.

6.    Yoshie H, Koushki N, Molter C, Siegel PM, Krishnan R, Ehrlicher AJ. High Throughput Traction Force 

       Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the 

       Epithelial-to-Mesenchymal Transition. J Vis Exp. 2019 Jun 1;(148):10.3791/59364.

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Department of Bioengineering

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