Review Cite This: Chem. Rev. 2018, 118, 6499−6515
pubs.acs.org/CR
Mechanobiology of Tumor Growth Parthiv Kant Chaudhuri,†,‡ Boon Chuan Low,*,†,§,∥ and Chwee Teck Lim*,†,⊥,# †
Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Level 9, Singapore 117411, Singapore Department of Biomedical Engineering, Columbia University, New York, New York 10027, United States § Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore ∥ University Scholars Programme, National University of Singapore, Singapore 138593, Singapore ⊥ Department of Biomedical Engineering, National University of Singapore, Singapore 117583, Singapore # Biomedical Institute for Global Health Research and Technology (BIGHEART), National University of Singapore, Singapore 117599, Singapore
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‡
ABSTRACT: Over the past decade, researchers have highlighted the importance of mechanical cues of the metastatic niche such as matrix stiffness, topography, mechanical stresses, and deformation on cells in influencing tumor growth and proliferation. Understanding the cellular and molecular basis and fine-tuning the mechano-response of cancer cells to this niche could lead to new and novel therapeutic interventions. In this review, we discuss the importance of mechanical cues surrounding tumor microenvironment that govern the growth and progression of cancer. We also highlight some emergent principles underlying the mechanosensing and mechanotransduction mechanisms that link cellular responses such as gene expression to phenotypic changes arising from such external cues. Recent technological advancements to visualize, quantify, model, and test these crucial steps with great precision will further advance our understanding of this phenomenon. We will conclude by showcasing potential applications of mechanobiology in controlling cancer growth as alternative cancer treatment regimes.
CONTENTS 1. Introduction Cancer Metastasis and Dynamic Tumor Microenvironment 2. Mechanical Cues Regulating Cancer Growth 2.1. Matrix Stiffness 2.2. Topography 2.3. Compressive Stress 2.4. Shear Stress 2.5. Mechanical Stretching 3. Mechanisms of Cancer Chemo-Mechanical Signaling 3.1. Cell Surface Receptors and Ion Channels 3.2. Actin and Microtubules As Bridges for Mechanotransduction 3.3. Nuclear Mechanics 4. Recent Technological Advancements to Study the Effect of Mechanical Cues on Cells and Tissues 4.1. Controlled Microenvironment 4.2. Cell Mechanics 4.3. Imaging 5. Concluding Remarks Author Information Corresponding Authors ORCID © 2018 American Chemical Society
Notes Biographies Acknowledgments References
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1. INTRODUCTION Cancer Metastasis and Dynamic Tumor Microenvironment
During the process of metastasis, cancer cells can spread from the primary tumor to specific distant secondary locations through the blood or lymphatic circulatory system. The secondary location for metastasis is restricted to specific target organs for particular types of cancer. For instance, breast cancer preferentially metastasizes to bone, brain, liver, and lung.1 However, for other cancers such as prostate cancer, metastasis happens only to bone.2 The reason for this specific choice of secondary niche has been attributed to the “seed and soil hypothesis” by Stephen Paget and the “mechanical hypothesis” by James Ewing. According to the “seed and soil hypothesis”, the cancer cells (“seed”) selectively grow and proliferate in a microenvironment (“soil”) that is conducive for its growth.3 The “mechanical hypothesis” suggests that the circulatory pattern of blood flow dictates the specific secondary niche.4
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Received: January 18, 2018 Published: June 21, 2018 6499
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Figure 1. Mechanical landscape of metastasis. Schematic diagram showing the effects of various mechanical cues during metastasis. In the primary tumor, the cells are exposed to compressive stress from the surrounding microenvironment and mechanical stretching due to the hyper proliferation of the cancer cells. Additionally, the extracellular matrix stiffens and arranges themselves in an anisotropic orientation due to the activity of the stromal cells such as cancer-associated fibroblasts and macrophages, which facilitate the invasion of the breast cancer cells toward the blood circulatory system. In circulation, the single circulating tumor cells (CTCs) and tumor microemboli are exposed to shear stress and are subsequently arrested in the narrow blood capillaries, where they undergo extravasation to form micrometastases at specific secondary niches such as bone, brain, liver, and lung.
Figure 2. Mechanical cues affect cancer growth. Stiffness: On soft substrates, cells spread less and have short actin filaments. However, on stiff substrates cells undertake a polarized morphology and develop stress fibers. Tumor cells preferentially show higher proliferation on matrix rigidity corresponding to their secondary locations, as discussed in the text. Reproduced with permission from ref 49. Copyright 2015 Springer Nature. Topography: During the progression of breast cancer metastasis, the ECM fibers arrange themselves in a parallel anisotropic orientation (TACS) and this helps in the invasion of the cancer cells away from the primary tumor with greater velocity and persistence. The anisotropic cues inhibit the proliferation of noncancer cells; however, the malignant cells evade these growth inhibitory cues and continue their uncontrolled proliferation. Stretching: Due to abnormalities in the tumor vasculature, tumor interstitial fluid pressure is greater in most solid tumors and it generates hypertension and mechanical stretching of the tumor cortex to increase cancer cell proliferation. Confinement: CTCs may get trapped within the narrow capillaries of the circulatory system and acquire an elongated morphology, which results in impaired cell division. Shear stress: Within the circulatory system, cancer cells are exposed to shear stress, and it triggers cell cycle arrest via the activation of integrin receptors. WBC, white blood cells; RBC, red blood cells.
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mechanical stretching
shear stress
compressive stress
topography
Matrix stiffness
mechanical cues
effect on growth
stretching increases proliferation stretching triggers re-entry of contact-inhibited cells into the S-phase of cell cycle
uniaxial stretching of 1 Hz
static stretching for 6 h
laminar shear stress of 1.2 dyn/cm2 shear stress of 15 dyn/cm2 shear stress of 12 dyn/cm2 tumor interstitial fluid pressure of 5 and 10 mmHg
confinement affects spindle formation and impairs cell division proliferation of the cells was not affected G1 arrest of cell cycle progression G2/M cell cycle blockage stretching increases proliferation
soft substrate decreased normal cell proliferation and induced apoptosis; transformed cells growth unaffected “tissue tropism” increased growth when the matrix stiffness is similar to that of the target organs where metastasis will occur cell proliferation increased on rigid substrates and vice versa rigidity dependent (cell growth depends on matrix stiffness); rigidity-independent (cell growth do not depend on matrix stiffness) MDA-MB-231 prefers rigid substrate and SKOV-3 prefers soft substrate mechanically-induced dormancy; anisotropic cues reduce MCF-10A proliferation; MDA-MB-231, MCF-7 proliferation unaffected ‘mechanically-induced dormancy through depth sensing’; matrix depths reduce MCF-10A, MDA-MB-231, and MCF-7 proliferation MDA-MB-231 and MCF-7 proliferation not affected proliferation reduced on 400 nm; increased on 300 nm; apoptosis increased on 23 nm proliferation of smooth muscle cells but not HeLa decreased on the micropillars spheroids attain maximum diameter of 400 mm in 0.5% (w/v) agarose but 50 mm diameter for 1% (w/v) agarose stress decreased proliferation and induced apoptosis mechanical stress impairs mitotic progression stress slows down tumor evolution but triggers cell invasion stress enhances the migration potential independent of any changes in cell proliferation
tubular confinement of cells inside 3D rolled-up, transparent nanomembranes
pressing cells against a membrane surface with a weighted piston
PDMS devices were designed to restrict tumor spheroids growth tumor spheroids are cultured in permeable elastic capsules
co-embedded single cancer cells with fluorescent microbeads in 2.0% (w/v) agarose gels
0.3−1% (w/v) agarose gel
PDMS substrates; micropillar diameter, length, and center-to-center distance of 3, 9, and 9 μm, respectively
polystyrene (PS) and poly-L-lactide (PLLA) substrates; gratings and square pits topographic features of 15 to 500 μm dimension poly lactic-co-glycolic (PLGA) films; hemispherical beads of width 23, 300, 400 nm
polydimethylsiloxane (PDMS) substrates; micropits pore width between 2−4 μm and depth between 1.5−9 μm
polydimethylsiloxane (PDMS) substrates; microgratings width between 2−4 μm
polyacrylamide substrates; Young’s modulus between 2.83−34.88 kPa
polyacrylamide gels; Young’s modulus between 0.15−19.2 kPa
polyacrylamide gels; Young’s modulus between 0.08 and 119 kPa
polyacrylamide gels; Young’s modulus between 0.6 and 3 kPa
polyacrylamide substrates; Young’s modulus between 4.7 and 14 kN/m2
experimental design
Table 1. List of Mechanical Cues Affecting Cancer Growth cell types
refs
colon cancer cells (RKO and HCT-8) colon cancer cells (SW480 and HT29) osteosarcoma cells (MG63) epithelial tumors (A431, A549) were transplanted into nude mice osteosarcoma cell lines MG-63, HOS/ MNNG, OST, U-2/OS, SaOS-2 mammary epithelial cells (MCF-10A)
mammary cancer (MCF7, 67NR, 4T1, and MDA-MB-231) and normal (MCF10A) cells HeLa cells
colon carcinoma cells (LS174T), mammary carcinoma cells (MCaIV), and myosarcoma cells (BA-HAN-1) nonmetastatic murine mammary carcinoma (67NR) cells colorectal cancer cell (HCT116) colon carcinoma cells (CT26)
13
50
44 45 12 47
11
39
37 38
36
35
34
31
A549 HeLa cells, smooth muscle cells
29
30
9
8,23
25
24
22
21
MDA-MB-231 and MCF-7
MCF-10A, MDA-MB-231, and MCF-7
MCF-10A, MDA-MB-231,and MCF-7
MDA-MB-231, SKOV-3
PC-3, mPanc96, A549, MDA-MB-231
single cell populations isolated from metastatic breast cancer cell line (MDA-MB-231) Glioma cell lines
normal and H-ras-transformed fibroblasts (NIH 3T3)
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These hypotheses are not mutually exclusive, and current studies suggest a role for both of them.5 Another interesting feature of metastasis is the temporal kinetics of the disease, such as breast and lung cancers colonize to similar secondary organs. However, breast cancer relapses after years or decades from first diagnosis,6 and for lung cancer, distant macro-metastases are established within a few months.7 Due to the hostile microenvironment in the secondary location, the disseminated tumor cells might lose their proliferation ability and enter dormancy for an indefinite time or they proliferate slowly into micrometastases as the proliferation rate balances with that of cell death. Therefore, the complex yet dynamic microenvironment that the cancer cells encounter during the course of their metastatic journey plays a critical role in deciding the final fate of the disease. Over the past decade, it has been observed that the mechanical cues of the tumor microenvironment including matrix stiffness,8 topography,9,10 confinement,11 shear stress,12 and mechanical stretching13 can dictate the behavior of cancer cells (Figure 1). For instance, highly metastatic melanoma cells (B16−F1) can be selected by culturing them in a soft fibrin matrix.14 The higher stiffness of the breast tumor microenvironment promotes malignant phenotype of the mammary epithelium by controlling the integrin expression and signaling.15,16 The extracellular matrix (ECM) can either favor cancer progression16 or induce invasive cells to behave in a healthy manner.17 Therefore, fine-tuning of the mechanical microenvironmental cues is essential in influencing cancer growth and development. In this review, we first highlight the mechanical cues of the tumor microenvironment that regulate the growth and proliferation of cancer cells (Figure 2, Table 1). We then decipher how these mechanical cues are transduced into specific biochemical responses (Figure 3) and describe recent technological advancements used in quantifying these parameters (Figure 4). We conclude by summarizing the potential applications of the field of mechanomedicine in treating cancer, for example, the ongoing clinical trials that use anticancer drugs to target the mechanotransduction machinery. Much has been discussed in various excellent reviews on the different biochemical environmental cues that affect cancer cell proliferation.18,19 However, few have offered detailed perspectives on the influence of mechanical cues on the development of tumor. Here, we undertake a comprehensive review on the literature over the past 10 years (2009−2018) in order to understand the influence of the mechanical cues from the cancer microenvironment on malignant growth, the chemomechanical signaling pathways involved in causing phenotypic changes, and the technological advancements to quantify such important parameters.
Figure 3. Mechanisms of mechanotransduction. Cell surface receptors and ion channels: cell senses the mechanical cues from the matrix such as stiffness, topography, stresses, and mechanical stretching using the integrin receptors and FA complexes. At the cell−cell adhesions, a cadherin−catenin-mediated adherens junction plays a crucial role in controlling force transmission across a monolayer of cell sheet. Additionally, several other cell membrane bound mechanoreceptors such as G-protein coupled receptors (GPCR), receptor tyrosine kinases (RTKs), and ion channels helps in sensing the mechanical niche. Cytoskeletal components: stress fibers are an important component of the cytoskeletal network and are composed of bundles of actin fibers and nonmuscle myosin II (NMMII) that generates contractile forces to sense the surrounding matrix. Nuclear dynamics and transcription factor shuttling: the nucleus is subjected to considerable mechanical tension by the surrounding cytoskeleton, and it senses the mechanical cues via the LINC complex, 3D chromosome organization, and nucleus to cytoplasmic shuttling of transcription coactivators such as YAP/TAZ.
responsive to these growth inhibitory mechanical cues and continue their uncontrolled proliferation.21 In another study, the concept of “tissue tropism” was observed with respect to rigidity sensing mechanism of cancer cells. Tumor cells preferentially show greater proliferation and malignant behavior when the matrix rigidity corresponds to that of the target organs where metastasis occurs.22 Breast cancer cells (MDAMB-231) metastasizing to bone prefer stiff matrix, whereas ovarian cancer cells (SKOV-3) that metastasize to soft omentum fat pad prefer more compliant substrate. Recently, actomyosin contractility was understood to play a crucial role in determining tissue tropism.8,23 On soft substrates, ovarian cancer cells exert higher traction forces and undergo epithelialto-mesenchymal transition (EMT).23 In the case of glioblastoma multiforme tumor cells that are stiffer than healthy parenchyma, cells show increased proliferation with formation of prominent stress fibers and focal adhesions (FAs) on the rigid matrix.24 On the basis of the proliferative response of different cancer cells lines on substrates of varying rigidity, it is believed that rigidity sensing is an intrinsic property of the individual cell line. Cells are classified as “rigidity dependent” when cell growth is regulated by the matrix stiffness (e.g., MDA-MB-231) and “rigidity independent”, where cells proliferate at the same rate regardless of the rigidity of the
2. MECHANICAL CUES REGULATING CANCER GROWTH 2.1. Matrix Stiffness
Mammary matrix density and stiffness are observed to increase in breast cancer patients as compared to that of a healthy person. This is mainly due to greater collagen cross-linking by lysyl oxidase. The higher stiffness of the breast epithelium is correlated with 4 to 6 times higher risk of developing breast tumor.20 In healthy fibroblasts and oncogenic H-Ras-transformed fibroblasts that show malignant characteristics, soft matrix is found to inhibit the growth of healthy cells and induced apoptosis. However, transformed cells are not 6502
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Figure 4. Technological advancements to study the effects of mechanical cues. Controlled microenvironment: (I) Micron scale topographic features with specific dimensions can be fabricated to mimic the architecture and orientation of the ECM in vivo. Microgratings provide anisotropic cues and cells preferentially align along the parallel direction of the grating axis. Reproduced with permission from ref 9. Copyright 2016 Springer Nature. Cells extend actin-rich protrusions such as invadopodia to sense the depth of the micropits. Reproduced from ref 30. Copyright 2017 American Chemical Society. (II) Micropillar arrays are used to vary the stiffness of the matrix by changing the height of the pillars at a constant diameter and to monitor the forces exerted by the cell at the individual adhesion sites. Cellular mechanics: (III) schematic illustration of the microfluidic device made of PDMS (polydimethylsiloxane) showing single-cell mechanophenotyping. Cells are introduced into the device under continuous flow, and the deformability of the cells are measured in real-time and correlated to their malignant property (close up). Reproduced with permission from ref 160. Copyright 2012 National Academy of Sciences. Imaging techniques: (IV) In order to image interior tissues in vivo using intravital microscopy, several optical window chambers have been established to optically access large areas without surgery. This includes the dorsal skin-fold chamber for investigating subcutaneous tumors, the cranial imaging window to study brain tumors, the abdominal imaging window to monitor intestine, pancreas, and liver tumors, and the mammary imaging window to visualize breast tumors. The mammary imaging window is used to image Dendra2-expressing breast tumor, and the color of around 100 cancer cells in highly vascularized region of the tumor were photoconverted from green to red using violet light. The locomotion of these cells was monitored over a period of time. It was observed that cancer cells with short hairpin RNA (shRNA)mediated knockdown of neural Wiskott−Aldrich syndrome protein (N-WASP) that mediate actin reorganization showed lesser movement away from the imaging site (bottom) as compared to control shRNA cells (top). This indicates that N-WASP is required for the intravasation of cancer cells in the primary tumor. Scale bar 70 μm. Reproduced with permission from ref 161. Copyright 2012 Company of Biologists Ltd.
matrix (e.g., PC-3).25 Therefore, the proliferation of cancer cells on matrices of different rigidity is dependent on the particular types of cancer, and cellular contractility plays a key role in determining this response.16,25 However, most of the rigiditysensing studies did not take into account the possible effect of soluble factors secreted by the stromal cells that are also an integral part of the tumor niche. Future studies should combine the effect of both biochemical and mechanical cues on the proliferative response of the cells to better understand the in vivo complexity.
proliferation of noncancer breast epithelial cells (MCF-10A) and induce a temporary dormancy via activation of actomyosin contractility. However, the malignant breast cancer cells (MDA-MB-231 and MCF-7) somehow bypass these growth inhibitory topographic cues and continue their uncontrolled proliferation.9 In another study, topographic features comprising gratings and square pits with dimensions of 15 to 500 μm did not affect the proliferation of MDA-MB-231 and MCF-7, and this may be because the cancer cells are less responsive to such large structures (the size of the cell is around 20 μm).29 Bone, which is composed of a porous matrix, is one of the principal locations of breast cancer metastasis. We examined the effect of the dynamic mechanical niche of the bone on cancer cell proliferation using well-defined topographic features with micrometre scale pits (known as micropits) of varying width and depth to mimic the physiological porous microenvironment in vitro. Our findings further support the notion of mechanically-induced dormancy through depth sensing, where cells could sense the different depths of the ECM using actin-enriched protrusions such as invadopodia and transduce a mechanical cue to regulate the contractile machinery, thereby reducing proliferation.30 When lung cancer cells were cultured on nanotopography surfaces (such as, nanosmooth, 23, 300, and 400 nm hemispherical surface features) but similar surface chemistry, proliferation was observed to increase on the 300
2.2. Topography
The organization and topography of the collagen ECM fibers in the mammary tissue undergo dynamic changes (tumorassociated collagen signature; TACS) during the progression of the metastatic disease.26 In a healthy individual, the collagen fibers are arranged in a random isotropic arrangement (TACS1); however, during tumor advancement, the fibers arrange themselves in a parallel anisotropic orientation (TACS-3).27 TACS-3 topography of the ECM fibers facilitates the migration of the breast cancer cells away from the primary tumor with greater persistence and velocity.28 In our recent work, we introduced the concept of mechanically-induced dormancy, whereby anisotropic cues provided by the aligned collagen fibers of the primary breast tumor environment can reduce the 6503
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narrow capillaries, the cancer cells divide and acquire an elongated cylindrical shape. Such tubular confinement of the cell affects the morphology and kinetics of the spindle formation, thereby resulting in impaired cell division.11
nm surface but reduce on the 400 nm surface and 23 nm surface-induced apoptosis.31 During invasion through the dense matrix, cancer cells squeeze and deform through the narrow pores of the tumor stroma. The nucleus is one of the largest organelles in the cell. When the cell passes through subnuclear length confinements, the nucleus can limit the invasion rate and be exposed to great deformation and conformational changes that may affect the mechanotransduction processes and cell behaviors.32,33 Micropillar topographies were used to study the effect of nuclear deformation on cell proliferation, and it was observed that the proliferation of healthy cells but not cervical cancer cells (HeLa) which decreased on such micro features. Healthy cells have a greater nuclear lamina and higher expression of lamin A/C compared to that of cancer cells, which may provide nuclear mechanical resistance against deformation.34 It will be interesting to investigate the role of the cell surface receptors and the cytoskeletal network in transducing these mechanical cues and causing proliferation reduction in healthy cells but not in cancer cells. Despite most of the in vitro studies mentioned above investigated the effect of topographic cues using well-defined homogeneous surface features, future efforts should focus on understanding the effects of complex heterogeneity of the matrix architecture in regulating cell behavior.
2.4. Shear Stress
Under in vivo condition, cancer cells can be exposed to stress generated by the interstitial flow within the tumor niche and stress produced by the blood flow within the circulatory system. Fluid shear stress generated by blood flow ranges from 0.5−4.0 dyn/cm2 in the venous circulation and 4.0−30.0 dyn/cm2 in the arterial circulation.43 When colon cancer cells were exposed to laminar shear stress of 1.2 dyn/cm2, proliferation of the cells was not affected under shear stress and static condition.44 However, shear stress of 15 dyn/cm2 for 24 h induced G1 arrest of colon cancer cell cycle progression via the activation of α6β4 integrins.45 Interestingly, in human osteosarcoma cells, 12 dyn/cm2 of shear stress induced G2/M cell cycle blockage by the activation of αvβ3 and β1 integrins.12 Therefore, shear stress affects cancer cell cycle progression via the activation of integrin receptors, and this mechanotransduction process is dependent on particular cancer types and magnitude of shear stress applied. It would be interesting to explore the effect of shear stress under similar experimental conditions using multiple cancer cell lines and clinical samples such as CTCs that are exposed to fluid shear inside the body.
2.3. Compressive Stress
2.5. Mechanical Stretching
Due to uncontrolled proliferation and growth of cancer cells, tumor exerts pressure on the ECM and neighboring tissues, which in turn exerts compressive forces or stresses on the tumor. When human colon carcinoma spheroids were cultured in agarose gel, the spheroids grew to a maximum diameter of 400 mm in 0.5% (w/v) agarose, but when compression was increased for 1% (w/v) agarose, only 50 mm spheroids was formed.35 Such growth inhibition of tumor spheroids along with increasing concentration of agarose gel might be attributed to increased cell packing, reduced proliferation, and triggering of compression induced apoptosis via the mitochondrial pathway in the regions of high solid stress.36 In addition to the greater compressive forces, higher concentration of agarose may alter other factors within the microenvironment such as the stiffness, pore size, and transport of nutrients that can also have an effect on the cell growth and possibly be taken into consideration. Tumor growth associated mechanical compressive stress induces a mitotic arrest in a cell population of the spheroid by impairing bipolar spindle formation during mitosis.37 Although compressive stresses have a beneficial effect of reducing tumor growth, it might also have a harmful consequence in triggering peripheral cancer cell invasion by promoting the formation of leader cells and increasing cellmatrix adhesions.38,39 Unlike other mechanical parameters, quantification of solid stress and elastic energy within a tumor is challenging. Recently, 2D spatial map of solid stress in excised or in situ tumors can be measured by monitoring the stressinduced tissue deformation after the solid stress is released in a regulated manner. With the use of this technique to map the solid stress in primary and metastatic tumor, it was observed that solid stress depends on the particular cancer cells and their microenvironment and solid stress increases as the tumor gets larger.40 While metastasizing through the vasculature, cancer cells might get trapped within the narrow capillaries, where they proliferate to form early micrometastatic colonies before leaving the circulatory system.41,42 Within the confinement of the
Tumor interstitial fluid pressure is higher in most solid tumors and is due to the abnormalities in the lymphatic circulation and tumor vasculature.46 Increased interstitial fluid pressure and compressive force produced by proliferating cancer cells might lead to permanent mechanical stretching of the tumor cortex.47,48 The mechanical stretching induced by interstitial fluid pressure results in increased proliferation of cancer cells.47 Static stretching for 6 h at an applied air pressure of 2 bar triggers the activation of YAP (yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) and reentry of contact-inhibited human mammary epithelial cells into the S-phase of cell cycle.13 This study highlights that mechanical forces regulate local YAP/TAZ activity. In the region of low mechanical stress at the center of the contact inhibited cells, YAP/TAZ is inhibited by F-actin-capping and severing proteins such as CapZ and Cofilin. However, the cells at the edges of the same multicellular sheet show YAP/TAZmediated proliferation that is regulated by actomyosin contractility. In fact, cell mechanics regulate YAP/TAZ activity in a distinct manner from Hippo pathway-induced YAP/TAZ phosphorylation and inhibition.13 Thus, mechanical stretching can overcome the growth arrest induced by contact inhibition via the activation and nuclear translocation of YAP/TAZ and serve as an initiator of tumor growth and proliferation.
3. MECHANISMS OF CANCER CHEMO-MECHANICAL SIGNALING 3.1. Cell Surface Receptors and Ion Channels
The progression of a tumor to a highly invasive, metastatic phenotype requires the interaction of the cells with various parameters of the ECM such as composition, stiffness, and architecture. Mounting evidence suggests that such physical cues can be sensed by the integrin receptors and transduced into biochemical signaling events to promote cancer metastasis and has been extensively reviewed elsewhere.51 Integrins are 6504
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heterodimeric transmembrane receptors composed of α and β subunits and there are 24 different types of integrin receptors in mammals that recognize different ECM ligands.52 The effect of ECM stiffness on cancer cell phenotype is dependent on the composition of the matrix and the type of integrin receptors involved to sense it.53 At the cell-matrix adhesion, integrins connect the matrix with the cytoskeletal network and transduce physical forces produced by the actin retrograde flow and actomyosin contractility to the ECM via mechanosensitive FA components known as “molecular clutches”.51 FAs are large macromolecular structures through which mechanical tension and regulatory cues are transduced between the matrix and the cell. At the early stages of cell adhesion, small (0.25 μm2) FAs known as focal complexes are formed at the leading edge of the cell in the lamellipodia, and they comprise integrin, talin, paxilin, vinculin, and actin.54,55 Focal complexes mature into stable FAs by recruiting zyxin.56 The molecular clutch senses ECM stiffness via mechanically induced protein conformational changes. Talin is a cytoplasmic adapter protein that connects the integrin to the actin. Talin has unique mechanosensory characteristics: first, talin autoinhibition restricts the protein in an inactive state. Subsequently, when the integrins are activated, talin forms a weak bond with the integrins that is essential for the formation of early adhesive structures such as focal complexes.57,58 The next level of regulation involves the mechano-activated opening of the vinculin binding sites within the talin rod domain. On rigid matrix, high mechanical forces unfold talin to expose the vinculin-binding sites, which results in vinculin binding and clutch reinforcement. However, on complaint matrix, low mechanical forces on talin are insufficient to induce vinculin-dependent force transmission.59 Therefore, talin plays a key role in FA maturation and transducing mechanical cues into biochemical responses by exposing the vinculin binding sites in a mechano-controlled manner. Cell-cell adhesion plays a crucial role in maintaining the integrity of a tissue, and they relay tension across groups of cells to regulate collective cell behavior. On substrates with tumorlike stiffness, mammary epithelial cells undergo EMT via the activation of transcription factors such as Twist.60,61 When two or more mammary acini with premalignant lesions are located in close proximity, radially aligned tracks of collagen form a superhighway between them that accelerates acini disorganization rate compared to single acini.62 Therefore, long distance mechanical communication between two premalignant acini increases the transition to a more invasive morphology. However, the role of chemical gradients formed between the two acini is not ruled out, and it could play a synergic role along with the collagen super highways to cause mammary structure disorganization. During EMT, cancer cells can acquire increased migratory potential and the force transmission at the cell−cell adhesion is altered by down regulation of E-cadherin mediated adherens junction. Occurrence of cadherin switching (switch from expressing E-cadherin to N-cadherin) is accompanied by redistribution of forces from adherens junction to cell-matrix adhesions.63 Therefore, understanding the molecular mechanisms of force transmission at such junctions is of utmost relevance in the context of EMT and cancer progression. Mechanical tension-induced conformational changes of αcatenin play an important role in controlling force transmission at the cadherin-catenin adhesion junctions. Indeed, α-catenin serves as a mechanosensor by regulating force-dependent binding with F-actin64 and vinculin.65 Cadherins and integrins
are linked to the actin cytoskeleton, and several studies suggest that mechanical tension driven crosstalk between them is essential for force transduction.66 Vinculin is one of the key molecules that are recruited to both the cell-matrix and cell− cell adhesion in a force-dependent manner, and it facilitates the reinforcement of the junctions. Interestingly, vinculin is phosphorylated at different tyrosine residues at the respective adhesions.67,68 Therefore, spatial regulation of vinculin phosphorylation plays a crucial role in determining its mechanical effect at the respective junctions. In addition to integrins and adherens junction, immunoglobulin (Ig) superfamily forms a key representative of cell adhesion molecules (CAMs). L1 cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (N-CAM) are crucial members of Ig-CAM family and participates in cancer cell proliferation, invasion, and metastasis. The expression pattern of L1-CAM and N-CAM is highly heterogeneous in different cancer types and have been correlated with patient overall survival.69,70 Since both L1-CAM and N-CAM are either directly or indirectly linked with the actin cytoskeleton and their expression varies with the metastatic stages of the disease, force transmission through these adhesion molecules might be an interesting topic for future investigation in the context of tumor mechanobiology.71 Apart from the integrin and cadherin receptors, several other cell-surface receptors such as G-protein coupled receptors, receptor tyrosine kinases, ion channels, and glycocalyx have emerged recently and have been considerably reviewed elsewhere.72 Among them, the mechanosensitive transient receptor potential vanilloid 4 (TRPV4) channels have gained particular attention. Mechanical cues can activate the channels to cause calcium influx and actin reorganization.73 Recently, it has been observed that lower expression of TRPV4 induces Rho/ROCK signaling pathways in tumor endothelial cells (TEC), which results in abnormal mechanosensitivity and angiogenesis.74,75 Interestingly, pharmacological activation of TRPV4 reduces TEC proliferation in vivo and improves cancer therapy. This study signifies the role of TRPV4 channels in controlling tumor angiogenesis via selective inhibition of TEC proliferation.76 3.2. Actin and Microtubules As Bridges for Mechanotransduction
Within a three-dimensional matrix, cancer cells reorganize cytoskeletal elements such as actin, microtubules, and intermediate filaments to adapt to the surrounding physical cues. Stress fibers are an important component of the cytoskeletal network, and each fiber is composed of bundles of actin fibers cross-linked by α-actinin, fascin, filamin, and zyxin. Nonmuscle myosin II (NMMII) is often, but not always, present in the stress fibers and is responsible for generating contractile forces.77 Stress fibers are also regarded as force transmitting structures because the tension within the stress fibers can be directly transmitted to the matrix through the FAs to generate isometric tension, and such changes in tension help the cell to sense surrounding forces.78 Stiff matrix provides resistance to cellular contractility and increases proliferation by generating higher Rho-ROCK-Myosin-based contractile forces, integrin clustering, and ERK activation.24 Anisotropic cues9 and depth sensing cues30 could activate actomyosin contractility and reduce the proliferation of MCF-10A cells. Interestingly, in response to depth-sensing cues, contractility is inhibited in MDA-MB-231 to reduce proliferation, which indicates that depending on the metastatic potential (MCF-10A vs MDA6505
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rounded nucleus was observed.88 The nucleus has a nonrandom spatial orientation of chromosome territories to carry out regulated transcriptional activation of genes.89,90 Recent evidence suggest that the relative orientation of the chromosomes, which are regulated by actomyosin contractile forces could have a greater effect on genome regulation.91,92 Moreover, substrate geometry can also control the shuttling of the transcriptional cofactors between the nucleus and the cytoplasm and its interaction with the target genes in the 3D chromosome organization.93 For instance, on rectangular patterns, chromosome 3 was located in the interior of the nucleus, whereas for circle patterns, it remained at the periphery of the nucleus.94 This particular positioning was correlated with the higher expression of myocardin-related transcription factor-A (MRTF-A)/serum response factor genes on this chromosome. Therefore, both 3D chromosome organization and nucleus to cytoplasmic shuttling of transcription factors can control gene expressions in response to mechanical cues. Mechanical cues such as matrix stiffness, topography, and cell geometry can alter the epigenetic regulation of gene expression via chromatin remodelling, noncoding RNA activity, and posttranslational modifications of histones.95−98 Cells seeded on stiff matrix display a transcriptionally active euchromatin, whereas soft substrate leads to a chromatin shift toward a transcriptionally inactive heterochromatin state.99 Histones are nucleoproteins that are responsible for packing the DNA within the nucleus into structural complexes called nucleosomes. Posttranslational modifications of histones such as by lysine acetylation, arginine methylation, lysine methylation, and serine phosphorylation play a crucial role in controlling gene expression. Recently, it has been observed that the rigidity of the hydrogel could influence the DNA methylation pattern and induce a sustainable mechanical memory to stabilize the global histone configurations.100 One of the crucial mechanism by which cells respond to matrix topography is the variation in the level of histone acetylation. Microgroove substrate induces histone acetylation and higher transcriptional activity compared to unpatterned control for different cell types such as mesenchymal stem cells,101 fibroblasts,102 and cardiomyocytes.103 Due to uncontrolled growth, tumor generates mechanical compression forces that trigger chemo-mechanical signaling pathways in cancer and stromal cells to promote malignant phenotypes. Compression induces downregulation of microRNA-9 via DNMT3A-dependent promoter methylation in breast cancer cell lines and cancer-associated fibroblasts that promotes vascular endothelial growth factor secretion and tumor angiogenesis.104 Interestingly, decompression could reverse compression-induced microRNA-9 downregulation and function of microRNA-9 target genes. These findings demonstrate that mechanical regulation of microRNA activities plays a critical role during tumor angiogenesis. The control of cell spreading and cell shape by the matrix can also influence chromatin structure and gene expression profile. 3D culture of human mammary epithelial cell induces histone deacetylation and decreased transcription.105 Intriguingly, this effect can be replicated by forced cell rounding or by reducing actin polymerization, which indicates that a complex combination of contractility-related signaling can regulate histone acetylation pattern depending on cell geometry.106,107 Therefore, the ability to regulate the epigenetic landscape using mechanical cues, either singly or in combination with other conventional methods, could provide a wealth of information to control
MB-231), cells adapt to an identical microenvironmental cue (depth of the matrix) using different signaling mechanisms.30 Recent studies have shown that the actin cytoskeleton could also act as a large-scale mechanosensor.49,79 As the matrix stiffness increases, the actin fibers tend to be more ordered and the number of stress fibers also increases.49,80 In addition to the organization of the fibers, the rheology of the actin could also be a possible mechanism for large-scale mechanosensing.49 On complaint substrate, the actin fibers are not strongly attached to the substrate, and this results in centripetal flow of actin toward the nucleus. In contrast, on the stiff substrate, the stress fibers are more stable and anchored firmly to the matrix. Microtubules are highly dynamic structures and undergo rapid remodelling by stochastic switching between polymerization and depolymerization phases. Previously, it was observed that microtubules prevented FA growth by suppressing contractility and localizing at the FA sites.81 Microtubules provide specific tracks for the transport of cargoes to and from FAs. Moreover, MT1-MMP (membrane type 1 matrix metalloprotease) requires microtubules and kinesin motors for surface localization to facilitate matrix degradation.82 Therefore, microtubules could favor the transport of MMPs to the FAs to degrade the interaction of integrins with the matrix and thereby inducing FA disassembly. 3.3. Nuclear Mechanics
The nucleus is the largest and stiffest organelle in the cell and is subjected to considerable mechanical tension by the surrounding cytoskeleton during cancer invasion through the constrictions of the 3D-matrix. The nuclear envelope (NE) plays a crucial role in sensing mechanical cues and adapting the morphology and function of the nucleus. NE is a double membrane bound structure and associated with subnuclear structures such as the linker of the nucleoskeleton and cytoskeleton (LINC) complex, the nuclear pore complex, and the lamina. Spanning the NE is the LINC complex that comprises Nesprin and SUN (Sad1 Unc-84) proteins. LINC complex provides a direct physical connection between the cytoplasm and the nucleoplasm that could facilitate the transduction of mechanical cues from the ECM. In the outer NE, Nesprins interact with the cytoskeletal network and with SUN proteins within the perinuclear region. In the inner NE, SUN proteins bind with lamins and nucleoplasmic constituents.83,84 The LINC complex resembles integrin-based adhesions in many respect.85 In both cases, a transmembrane protein (integrin or Nesprin-SUN complex) passes through the lipid bilayer and transduce forces between the two compartments through binding with the cytoskeletal filaments on one side and fibrous protein (ECM or nuclear matrix) on the other side. Similar to integrin-based clustering, LINC complex also forms clusters known as transmembrane actin-associated nuclear lines that allows force transmission across the NE for proper nuclear positioning.86 Apart from its structural similarities, LINC complex can act like cell surface adhesions and undergo remodelling in response to forces to strengthen the communication between the cytosol and the nuclear lamina.87 Substrate geometry can regulate the cytoskeleton-mediated stresses and morphology of the nucleus. Two extreme geometries such as, the anisotropic rectangles and the isotropic circles were micro-patterned on cell culture dishes. In rectangle shapes, cells develop strong stress fibers and a flattened nucleus. However, in circle patterns, short filaments of actin and a 6506
DOI: 10.1021/acs.chemrev.8b00042 Chem. Rev. 2018, 118, 6499−6515
Chemical Reviews
Review
stromal cells such as endothelial cells, fibroblasts, immune cells, and mesenchymal stem cells (MSCs) has recently been extensively reviewed elsewhere.123,124 Microfluidics technology has provided an opportunity to replicate such intricate cell−cell communications in vitro with high spatiotemporal resolution and a dynamic three-dimensional microenvironment. The transition of mammary epithelial cells to their invasive phenotypes was studied by coculturing cancer cells with fibroblasts in a microfluidics device that provides a costeffective solution to screen for the inhibitors of various pathways involved in this transition process.125 Latest advancements in tissue engineering, microfluidics, and biomaterials have led to the development of organ-on-a-chip and tumor-ona-chip platforms that can precisely regulate organ scale complexity and 3D microenvironment under dynamic flow conditions.126,127 Such devices need to be further developed to replicate the multifaceted chemo-mechanical coupling in the tumor microenvironment in vitro and to understand the cellular responses to biochemical cues in the mechanical context.128
desired cellular responses during reprogramming and diseased state.96 Recent studies have shown that translocation of YAP/TAZ between nucleus and cytosol can serve as a key sensor of mechanical cues in the cancer microenvironment and have been extensively reviewed elsewhere.108,109 Cyclic stretching of cells on soft substrates can mimic that of a rigid matrix in promoting cell growth and stress fiber formation. Furthermore, on application of cyclic stretch, MRTF-A translocate into the nucleus at a faster rate compared to YAP to enhance proliferation.110 In order to undergo successful metastasis, cancer cells need to detach from the matrix and escape anoikis during their journey through the blood circulation. Cell adhesion to the matrix controls YAP activity. During cell attachment, YAP is activated, whereas detached cell have inactivated YAP and undergoes anoikis.111 Interestingly, expression of constitutively active YAP facilitates the survival of detached cells, implying that cancer cells with activated YAP can metastasize efficiently. Recently, Rho GTPase activating protein 29 (ARHGAP29) was discovered as a transcriptional target of Yap in human gastric cancer cell lines.112 Intriguingly, Yap promotes the activity of ARHGAP29, and higher ARHGAP29 expression is positively correlated with metastatic potential and reduced overall survival of gastric cancer patients. Therefore, spatiotemporal activation of YAP in response to mechanical cues can overcome growth inhibition and anoikis induced by soft substrates and matrix detachment, which could be a crucial regulator of malignant growth.
4.2. Cell Mechanics
Quantitative understanding of how cancer cells respond to the mechanical cues requires technologies that can apply controlled forces on the cells and simultaneously measure the cellular morphology and changes in molecular pathways. Cancer cells are more deformable than healthy cells due to a disorganized and less filamentous cytoskeletal network as it helps the malignant cells to squeeze through the narrow constrictions of the ECM. Using microfluidic straight channels of specific dimensions, the deformability of healthy (MCF-10A) and nonmetastatic (MCF-7) breast cancer cells were quantified in terms of cell transit time, cell velocity, and cell deformation.129 Recently, microfluidics based deformability cytometry was developed to quantify the deformability at a single cell resolution in real time to diagnose clinical pleural effusion samples as benign or malignant with 91% sensitivity (Figure 4III).130 Several other powerful tools to quantify the local mechanical properties of cancer cells in vitro include atomic force microscopy, micropipette aspiration, and magnetic and optical tweezers has been thoroughly reviewed elsewhere.131,132 The force exerted by the cancer cells on the substrate can be quantified using 2D traction force microscopy (TFM). In this technique, the deformation of fiducial markers such as beads at the cell−matrix interface is imaged and computational algorithms are used to determine the contractile forces that caused the deformation. Using this technique, it was observed that cancer cells exerted higher contractile forces on rigid matrices and at the later stages of the metastatic disease.133 However, to understand the complex interplay between mechanical forces during cancer metastasis, 3D TFM134 is necessary to monitor the tension at the tissue level135 and at a physiologically relevant 3D matrix with higher spatiotemporal resolution.136 Subcellular nanosurgery can be used to measure tension inside a live cell or a tissue by focusing a laser beam to irradiate nano or microscale structures (such as individual stress fibers) within a sample without affecting its viability. The sensitivity of intracellular nanosurgery has been further improved to ablate a very small region within a cell (
