Are cancer cells really softer than normal cells

TL;DR: It is argued that cancer cells can indeed be considered as softer than normal cells, and the intracellular elements that could be responsible for the softening of cancer cells are focused on.

About: This article is published in Biology of the Cell.The article was published on 2017-05-01 and is currently open access. It has received 216 citations till now. The article focuses on the topics: Cancer cell & Cancer.

Summary

Submitted on 14 Mar 2017

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Summary

• Solid tumors are often first diagnosed by palpation, suggesting that the tumor is more rigid than its surrounding environment.
• Paradoxically, individual cancer cells appear to be softer than their healthy counterparts.
• The authors then focus on the intracellular elements that could be responsible for the softening of cancer cells.
• Most solid tumors are more rigid than their surrounding environment.
• The mechanical differences between normal and cancer cells could be primarily due to modifications in the cytoskeleton, but other factors, such as the cell microenvironment, internal membrane trafficking or non-equilibrium active forces, may contribute.

Contents

• It is still not clear whether cancer cells all have the same material properties with softening being a universal feature or whether mechanical forces and features depend on the tumor type (Jonietz, 2012).
• During the different steps of the disease, cancer cells acquire biological properties that cannot be found in normal cells.

Mechanics of cancer cells

• The ability of cancer cells to create metastases is certainly the main reason why cancer cells were thought to be different from healthy cells, not only from a biological point of view but also from a mechanical point of view (Mierke, 2013).
• Probing cellular rigidity at different scales A large number of experimental tools have been developed to measure the rheology of complex materials, also known as Microrheology of cancer cells.
• Visco-elastic materials show behaviors typical of both elastic solids and viscous fluids (Fig. 2C).
• To compare the quality of different models to describe the data, some recent studies have used more than one model in their analysis (Chan et al., 2015; Lim, Zhou, & Quek, 2006).
• Even if a relevant comparison of the mechanical properties of normal cells and cancer cells obviously requires comparing cells from the same organ, some AFM studies compared normal and cancer cells originating from different organs and also found that cancer cells are softer and more deformable.

The cell microenvironment

• For the last ten years, mounting evidence shows that the cell microenvironment influences cell behavior.
• In particular, cells sense the stiffness of the matrix (Lange & Fabry, 2013).
• The mechanical properties of the extracellular matrix (ECM) plays an important role in the metastatic process as stiffening of the ECM was shown to promote growth and invasion of mammary tissues both in vivo and in culture (Levental et al., 2009).

The cytoskeleton

• Several works comparing the stiffness of normal and cancer cells have thus investigated cytoskeletal organization in cancer cells and tried to correlate changes in the levels of expression of cytoskeletal proteins with the observed decrease in cell stiffness.
• Most studies have focused on the actin cytoskeleton, especially on the actin cortex at the plasma membrane and on actomyosin complexes, but interest in microtubules and intermediate filaments has also been growing rapidly in the past two decades.

Actin

• Modifications of the actin cytoskeleton during the metastatic process are well described (Nürnberg, Kitzing, & Grosse, 2011; Peckham, 2016; Vignjevic & Montagnac, 2008).
• Notably the relative amount of filamentous actin (F-actin) and monomeric actin (G-actin), which indicates the level of actin polymerization, depends on the cell malignancy.
• However conflicting results have been reported which seem to be cell-type dependent.
• More significantly, the organization of the actin cytoskeleton appears to be different in cancer cells.
• Similar observations were made in metastatic MDA-MB-231 breast cancer cells plated on adhesive micropatterns to standardize their intracellular organization when compared to non-tumorigenic MCF-10A cells, with changes in actin organization correlating with a strong decrease in cell stiffness in the metastatic cell line (Mandal et al., 2016).

Microtubules

• The role of microtubules in cell rigidity seems less clear than that of actin and, here again, could be cell-type dependent.
• A small but opposite effect of nocodazole was observed in retinal pigment epithelial (RPE-1) cells (Mandal et al., 2016) and in HeLa cells (Wilhelm, Gazeau, & Bacri, 2003), while microtubule stabilization by taxol induced a clear increase in stiffness in RPE-1 cells (Mandal et al., 2016).
• Even if overexpression of 3 tubulin correlates with tumor malignancy and resistance to chemotherapeutic drugs (Ferrandina et al., 2006), whether this is due to a change in cell mechanics has not been investigated.
• The amount of microtubules relative to actin filaments differs in different grades of colon cancer cells but this difference seems to be due to the total amount of actin (Pachenari et al., 2014).
• The organization of the microtubule network in three breast cancer cell lines with different metastatic potential did not show any significant variations (Calzado-Martín et al., 2016).

Intermediate filaments

• There are six types of intermediate filaments (IFs) differentially expressed depending on the cell type and the IF proteins that compose the filaments.
• Reducing the amount of vimentin in MDA-MB-231 cells decreases cell stiffness while overexpressing vimentin in MCF7 cells increases cell stiffness (C. Y. Liu et al., 2015) as expected if vimentin IFs contribute to cell stiffness.
• Downregulation of keratin during EMT may play a role in cancer cell softening.
• Even if vimentin and keratins are involved in cancer cell mechanics, their exact roles and how they are related are not entirely clear yet.
• Since IFs are heteropolymers and bundle within the cell, compensation mechanisms between the different IF proteins may play a role in mechanics when the expression of IF proteins is modified.

The nucleus

• At the scale of the whole cell, the nucleus is a major organelle contributing to cell mechanics (Zwerger, Ho, & Lammerding, 2011).
• As an example, the deformability of the nucleus is the limiting factor when cells migrate through tight constrictions (Lautscham et al., 2015; McGregor, Hsia, & Lammerding, 2016; Raab et al., 2016).
• In cancer cells, the nucleus morphology is altered compared to healthy cells (Denais & Lammerding, 2014).
• The volume of the nucleus is larger and its shape is more irregular.
• The expression of nuclear envelope proteins such as lamins is modified leading to breakage of the nuclear envelope (Bell & Lammerding, 2016; Davidson & Lammerding, 2014).

The plasma membrane

• The bending rigidity of the plasma membrane could contribute to the overall cell stiffness.
• The rigidity of the plasma membrane is controlled by its lipid composition and lipid synthesis and metabolism are known to be implicated in tumor development (Baenke, Peck, Miess, & Schulze, 2013).
• Supposing that fluctuations are of thermal origin, larger fluctuations indicate a lower rigidity in the highly metastatic cells.
• Interestingly, thermal noise is not the only source of membrane shape fluctuations and active non-equilibrium energy-dependent forces are thought to enhance such fluctuations (Betz, Lenz, Joanny, & Sykes, 2009; Boss et al., 2012; Manneville, 1999; Tuvia, Levin, Bitler, & Korenstein, 1998).
• It was shown for instance that the local intracellular stiffness increases in the proximity of the Golgi apparatus (Guet et al., 2014).

Signaling proteins

• The interplay between cellular rigidity and the signaling pathways associated with the cytoskeleton and membrane trafficking is complex.
• From a fundamental point of view, understanding the modifications in cell rigidity in cancer cells could help identifying which signaling pathways or proteins are involved during tumorigenesis.
• It has been shown that ROCK-dependent modifications of the keratin network play a role in cell stiffness (Bordeleau, Myrand Lapierre, Sheng, & Marceau, 2012).
• Another example is given by the adhesion protein E-cadherin, an epithelial marker which levels differ in healthy and cancer cells.
• Breast highly invasive MDA-MB-231 cancer cells do not express E-cadherin but express N-cadherin instead, while non-invasive MCF-7 and slightly invasive T470 cell lines exhibit E-cadherin levels that inversely correlate with their invasiveness (Omidvar et al., 2014).

Non-equilibrium active forces

• In recent years, the role of active forces, such as the forces exerted by ATP-consuming molecular motors, in cell mechanics has attracted a lot of attention.
• Recently, a new technique based on optical tweezers called Force Spectrum Microscopy has been introduced to measure the effect of fluctuating forces caused by active processes on the cytoplasm rheology (Guo et al., 2014).
• Most studies which have compared the mechanics of individual normal and cancer cells have found that cancer cells are softer than healthy cells.
• Associating several techniques, especially techniques operating at different scales, should help resolve this issue.
• Differences not only exist between tumors of the same type in different patients, but also between cells within a tumor.

Figure and table legends

• Figure 1 Experimental techniques used to compare the rheological properties of healthy and cancer cells at the scale of the whole cell (left) or at the local scale .
• In a creep (resp. stress relaxation) experiment a constant stress (resp. strain) is applied.
• The stress is proportional to the strain and the proportionality constant is the Young modulus E (Pa).
• The Standard Linear Liquid (SLL) model is composed of a Kelvin-Voigt element in series with a dashpot.
• Similarly, changes in the extracellular matrix (ECM) stiffness and of the expression levels of intermediate filament proteins, could explain changes in cell mechanics observed in cancer.

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