Cancer Mechanobiology: Microenvironmental Sensing and Metastasis

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Cancer Mechanobiology: Microenvironmental Sensing and Metastasis GeonHui Lee, Seong-Beom Han, Jung-Hwan Lee, Hae-Won Kim, and Dong-Hwee Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01230 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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ACS Biomaterials Science & Engineering

Cancer Mechanobiology: Microenvironmental Sensing and Metastasis

GeonHui Lee1,*, Seong-Beom Han1,*, Jung-Hwan Lee2,3, Hae-Won Kim2,3,4, and DongHwee Kim1,ǂ

1

KU-KIST Graduate School of Converging Science and Technology, Korea University,

Seoul, 02841, South Korea 2

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan

31116, South Korea 3

Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan


Department of Nanobiomedical Science & BK21 PLUS Global Research Center for

Regenerative Medicine, Dankook University, Cheonan 31116, South Korea

*Equally contributed to this work ǂ Author for correspondence ([email protected])

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ABSTRACT Cellular microenvironment plays an important role in regulating cancer progress. Cancer can physically and chemically remodel its surrounding extracellular matrix (ECM). Critical cellular behaviors such as recognition of matrix geometry and rigidity, cell polarization and motility, cytoskeletal reorganization, and proliferation can be changed as a consequence of these ECM alternations. Here, we present an overview of cancer mechanobiology in detail, focusing on cancer microenvironmental sensing of exogenous cues and quantification of cancer-substrate interactions. In addition, mechanics of metastasis classified with tumor progression will be discussed. Mechanism underlying cancer mechanosensation and tumor progression may provide new insights into therapeutic strategies to alleviate cancer malignancy.

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1. Introduction Cancer cells are exposed to a wide range of exogenous biochemical and physical signals from various sources. The majority of cancer research is based on biochemical mechanism since many types of cancer can be diagnosed with biomarkers based on cancer characteristics such as DNA copy number assessment, mutation screening, mRNA profiling, proteomic profiling, and metabolomics profiling 1-2. However, recent studies have further identified that many abnormal cells (e.g. cancer) can also be detected based on the difference of matrix rigidity 3. Such rigidity difference between tumor and normal tissue is induced by the extracellular matrix (ECM) remodeling during tumor development 4, increased population of cancer cells 5-6 and intrinsic stiffness change of cancer cells 7-8. Discovering the effect of various mechanical cues on cancer cell can provide better understanding of cancer and positional treatments. Using mechabiological approach to understand cancer behaviors based on the process of cellular sensation of mechanical and physical signals provided by the extracellular milieu is a growing field that has attracted recent interest along with biochemical approach. Indeed, mechanobiology in cancer research including tumor development, invasion and metastasis has received great attention to reveal the mechanism of cancer cell microenvironmental sensing and tumor progression. Dynamic microenvironment of cells such as stretching 9-10, compression 11, shear stress 12, matrix rigidity 13-14 and microenvironmental topography 15 can change the morphology, polarity, motility and fate of cancer cells 16-17

.

In this review, we will provide detailed description of cancer mechanobiology, including how external forces can affect cancer cells and quantitative measurement of interaction between cancer cell and substrate at single cell level. We will also describe diverse in vitro microplatforms to determine mechanosensation involved in proliferation, polarization, directing migration and invasion of cancer cells. In addition, we will discuss metastasis mechanics in various metastasis stages. Finally, we will briefly comment on therapeutic potential of cancer mechanobiology.

2. Mechanical properties of cancer cell Mechanical properties of cells are known to be related to diverse biological processes such as cell migration, proliferation, differentiation, and development

18

. They are also associated with many

diseases, including inflammation, laminopathies, cardiovascular disease, and cancer

19-23

. Cancer cells

can reorganize their cytoskeleton in different grades to promote invasion and enhance metastases. Recently, mechanical properties of cancer cells have been used as biomarkers to detect cancer in early stages24. To explore cell mechanics, various technologies including atomic force microscopy (AFM) 2534

, optical tweezer

27, 35-36

, magnetic tweezers

37

, optical stretcher

38-39

, micropipette aspiration

40-41

,

3



microfluidic cytometry stretcher

47-48

42-43

, magnetic twisting cytometry

44

, microrheology

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45-46

, and microplate

have been developed. We will introduce these tools and show mechanical properties of

different types of cancer (Table 1). AFM is common technology used to measure mechanical properties of adherent cells and non-adherent cells. The characteristic of AFM includes high sensitivity, high resolution, force measurement, real-time imaging, and well working in liquid environment which is essential for biological sample. When the cantilever scans the surface of a sample, the cantilever is deflected by forces between the sample and the tip. Laser light emitted from a photodiode is reflected and provides a quantitative signal to the detector as degree of cantilever deflection (d) for calculation using Hooke’s law (F = -kd) and calibrated spring constant value (k). Various studies have measured the Young’s modulus for cancer cells originated from different tissues (breast 26-29, prostatic 28-30, ovarian 31-32, bladder 29, 33, leukemia49) and normal cells using AFM. According to Q.S. Li et al., non-malignant (MCF-10A) human breast epithelial cells have higher (1.4 – 1.8 times) Young’s modulus than malignant MCF-7 cells at the same experimental condition, where actin stress fiber is well-organized in non-malignant cell lines 26. W Xu et al. have demonstrated that the elasticity of ovarian cancer decreases as the cancer is more aggressive comparing non-malignant ovarian cancer cell line (IOSE) and malignant ovarian cancer cell line (HEY, HEY A8 – more aggressive)

32

. Elasticity and viscoelasticity of malignant cancer cells (HEY -

0.884±0.529 kPa, HEY A8 - 0.494±0.222 kPa) are softer than non-malignant cancer (2.472±2.048 kPa) whereas their migration and invasiveness are higher. These results were also confirmed in primary cancer cells taken from patients

25, 28, 30

. M Lekka et al. have compared uterine corpus, breast, vulvar

cancer tissue sections and non-neoplastic tissues. In the case of uterine corpus, the stiffness of cancer regions (0.57±0.19 kPa) is lower than that of non-neoplastic region (1.27±0.13 kPa). Although AFM is a well-experimented method to measure physical properties of cells, it has the disadvantage in that it should have contact with sample directly, which could induce alteration of cellular properties. Optical tweezer is a non-invasive method that can measure mechanical properties of a single cell, where optical trap manipulates small dielectric particles using highly focused laser. Optical tweezer force can induce maximum 100 pN to particles ranging from 20 nanometers to several micrometers in size. Taking advantage of these fine control possibilities, optical tweezers have been used extensively to study protein and DNA molecules

50-51

, as well as mechanical properties of cells. Using optical tweezer,

various studies have demonstrated biophysical properties of different types of cells 52-55. For instance, D Cojoc group has identified the mechanical properties of breast cancer and normal epithelial cells using optical tweezer and AFM, respectively 27. They also showed that elasticity values measured by optical tweezer were more than 1000 times lower than those by AFM due to indentation force. Highly aggressive breast cancer cell line MDA-MB-231 (Optical tweezer: 12.6±6.1 Pa / AFM: 55.6±20.1 kPa) 4


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is the softest based on optical tweezer and AFM measurement whereas elasticities of normal cell HBL1000 (Optical tweezer - 23.5±10.6 Pa / AFM – 95.4±43.6 kPa) and luminal cancer MCF-7 (Optical tweezer – 30.2±15.0 Pa / AFM – 87.3±47.8 kPa) are similar in these two measurements. Furthermore, they investigated the cancer cell stiffness on PDMS substrate that is more compliant than glass substrate 36

. MDA-MB-231 cells are stiffer on PDMS than those on glass whereas HBL-1000 and MCF-7 cells

are softer. Cell spreading area that is normally larger on stiff substrate than that on soft substrate is increased when the substrate becomes soft in MDA-MB-231 cells. These results demonstrate that MDA-MB-231 cells are more affected by the surrounding physical environment than HBL-1000 and MCF-7 cells. Although optical tweezer is used to various studies, this technic has some drawbacks such as limited force measurement (maximum 100 pN), local heat accumulation by focused high intensity laser, and low selectivity. Magnetic tweezer is a physical technique to measure the mechanical response of cells using manipulate magnet beads with a magnetic field, where forces can be controlled to be ranging from piconewtons (pN) to nanonewtons (nN). Magnetic tweezer is commonly used to biological molecule studies such as proteins and DNA for single-molecular force measurements

56-57

. In the late 90s, several researchers

utilized this method to probe the mechanical response of cells using a bead coated by extracellular matrix to attach it at the cell surface 58. V Swaminathan et al., have demonstrated that the degree of invasiveness is related to mechanical deformability of cancer cells using a transwell assay and a 3D force magnetic tweezer in cell lines and primary cells 37. In human ovarian cancer cell lines, HEY is the most invasive (I HEY - 0.85%) whereas IGROV is the least invasive (I IGROV - 0.006%) in transwell assay. The mechanical properties of HEY are 10 times more deformable than IGROV. These results are consistent with primary cells, e.g., primary epithelial ovarian cancer cell OV207 was about 30 times more invasive, and about 10 times softer than OV445 37. Optical stretcher is a biophysical tool using dual-beam laser to trap suspension cells. In optical stretcher, photo momentum from the laser can be transferred to the cell surface and induce mechanical force to stretch and/or deform the cell, where force is applied in the direction of the optical axis. The optical stretcher is a non-invasive system that can be utilized in high-throughput screening in combination with a microfluidic system to measure properties of cells. Based on this scheme, J Guck’s group reported the deformability of breast cancer cells and normal cells using microfluidic optical stretcher 38 39. These results were consistent with those of AFM studies. Human breast epithelial MCF-10 cells were found to have the lowest optical deformability (10.5±0.8 %), followed by cancerous breast epithelial MCF7A cells (21.4±1.1%) and metastatic breast epithelial MDA-MB-231 cells (33.7±1.4 %). Furthermore, they measured the deformability of the modified cancer cells treated with chemicals that could make them more or less aggressive. In the case of the more aggressive modMCF-7 cells (30.4±1.8 %), they 5



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were deformed more severe than MCF-7 cells. While the best characteristic of the optical stretcher is that it can be developed as a fluidic system, laser can damage the cells and thus this issue should be resolved for more applications. Micropipette aspiration is one of general biophysical techniques for measuring viscoelastic properties of cells. A part of the cell or the whole cell is deformed by aspirating through a micropipette with negative pressure. Micropipette aspiration can exert a wide pressure range from pN/μm2 to nN/μm2. M Pachenari et al. have demonstrated the relationship between mechanical properties of cancer and cytoskeleton using micropipette aspiration in different grades of colon cancer cells 40. The elasticity of high-grade colon cancer (SW48 – 56.44 Pa) indicates about 0.7 times more deformable than low-grade colon cancer (HT29 – 82.31 Pa). By comparing cytoskeleton contents using western blot and fluorescence analysis in SW48 and HT29 cell lines, they found that the ratio of actin filaments to microtubules in HT29 (western blot: 1.98±0.20, fluorescence analysis: 2.48±0.85) were larger than SW48 (western blot: 1.48±0.26, fluorescence analysis: 1.33±0.45). These results presented that microtubules attained an effective role in gradual cytoskeletal reorganization while transforming from nonmalignant to malignant cancer. Recently, it has been confirmed that fluid shear stress can induce alteration of the mechanical property of cancer cells using micropipette aspiration 41. Non-transformed prostate epithelial cells (PrEC LH) were compared to transformed prostate cancer cells (PC-3) after they were exposed to shear stress or at unsheared state. PrEC LH cells (47.72±25.7 Pa) are stiffer than PC-3 cells (19.95±6.77 Pa) under non-sheared condition. After exposure to shear flow (6,400 dyn/cm2), the stiffness of PC-3 cells was increased by about 77% whereas that of PrEC LH cells was not changed. This result suggests that transformed cancer cells could adapt to fluid shear stress although underlying cellular mechanism responsible for the alteration of cell stiffness still remains unanswered 41. As witnessed, the measurement of the mechanical properties of cells confirmed that cancer cells have softer properties compared to normal cells. In addition, cancer cells have been demonstrated to have more deformable properties with higher invasive capacity. These results indicate that cytoskeleton formation of cancer cells is different from that of normal cells, which further suggests that physical properties of cancer cells play an important role in migration and metastasis.

3. Measurement of cell-substrate interaction force Cellular forces are essential in various biological processes, including cell migration, differentiation, adhesion, signaling, embryogenesis, and cancer metastasis. Intracellular forces propagate mainly through the cytoskeleton that generates mechanical stress via actomyosin contractility. Since altered cytoskeletal architecture of cancer cells induces different mechanical properties compared to normal 6


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cells, cellular tension is expected to be a biomarker together with the mechanical stiffness of cells to diagnose early stage cancers. This chapter will discuss various methods for measuring cellular tension in cellular and subcellular level and introduce cellular force characteristic of cancer cells. Gel-based traction force microscopy (TFM) is one of the most representative methods of measuring cellular forces generally ranging from nanonewtons (nNs) micronewtons (µNs) 59-65. In TFM, cellular forces are calculated by the magnitude of elastic gel deformations using fluorescent beads inside the hydrogel. Polyacrylamide hydrogel is generally synthesized to form 2D deformable substrate, which is coated with extracellular matrix proteins such as collagen and fibronectin to better adhere cells. When cells exert adhesion force to the substrate, position changes of the microbeads represent the gel deformation. This method facilitates diverse studies on cell-substrate interactions by simply controlling the stiffness of hydrogel substrate. CM Kraning-Rush et al. demonstrated that cellular traction forces of various cancer cell lines (breast, prostate, and lung) were larger than those of normal cell lines on PA hydrogel (5 kPa) 62. In addition, they found that cellular traction forces were increased with metastatic potential and transformed cells were more invasive. R Kristal-Muscal et al. further reported similar outcomes using the breast cell lines with different invasive potential on soft PA hydrogel substrate (2 kPa) 63. On the other hand, V Peschetola et al. showed that traction forces of two types of epithelial bladder cancers (invasive T24 cell line, less invasive RT112 cell line) were different on PA hydrogel of 10 kPa stiffness 64. They also measured the traction force of cell outliers and half-cell (leading edge) in migrating cells. Less invasive RT112s (traction stress: 171 Pa, traction force: 22.8 nN) displayed larger traction stress than more invasive T24 cells (traction stress: 120 Pa, traction force: 17.4 nN) although differences of traction force were not statistically significant. These results demonstrate that the invasive cancer cells generally have larger traction force than the non-invasive cancer cells. However, it has been recognized that not all invasive and non-invasive cancer cells follow these results. TM Koch group have investigated the relation between traction force and cancer invasion using 3D collagen gel. They cultured breast cancer cells (highly invasive: MDA-MB-231, non-invasive: MCF-7), lung cancer cell lines (highly invasive: A-125, non-invasive: A-519), and non-invasive vulva cancer cells (A-431) 65. Strain energy values of more metastatic cancer cells (2.86 pJ for A-125 and 0.78 pJ for MDA-MA-231) were larger than those of non-invasive cells (0.10 pJ for A-549 and 0.35 pJ for MCF-7) originated from the same organ. However, the traction force does not exactly reflect the invasive capacity of cancers cells in case that they originate from different tissues, e.g., traction force of highly invasive breast cancer cells (MDA-MB-231) is smaller than non-invasive vulva cancer cells (A-431) 65. The Micro-post array is another commonly utilized method for measuring cellular traction force

66-71

.

In micro-post array, cells are placed on top of microscale elastomeric post array and cellular traction forces are calculated by the magnitude of post deflection. Micro-post arrays are usually made by 7



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polydimethylsiloxane (PDMS) or silicon and their mechanical properties is dependent on post diameter, spacing between posts, and elastomeric stiffness. Previous studies have compared the magnitude of cellular traction force between cancer cell and normal cell. Z Li et al. have investigated cellular forces of three distinct cells (normal mammalian mechanocytes, benign tumor L929 cells, and malignant HeLa cells) using a silicon-nanowire post with diameter of 280 ± 40.4 nm and elastic modulus at 151 ± 38.3 GPa 70. Average cellular traction forces of L929 cells (3.48 ± 0.46 μN) and HeLa cells (2.84. ± 0.49 μN) are 1.5 and 1.22 times higher, respectively, than those of mechanocytes (2.32 ± 0.16 μN). In addition, they compared cellular traction forces of mechanocytes and L929 cells for 24 hours. The traction force of benign tumor L929 cells was increased from 1.77 to 6.2 μN, whereas that of normal mechanocytes was relatively unchanged. The increase of traction force follows the spreading area of L929 cell. These results indicate that cancer cells can exact large traction force to substrate. Recently, various groups demonstrated that traction force of breast cancer cells can change when they are treated with anti-cancer drug 71. After human breast cancer cell (MCF-7) and human normal breast epithelial cell (MCF-10A) were cultured on gallium phosphide nanowires with about 100 nm in diameter, the traction force of cancer cell was larger than normal epithelial cell without anti-cancer drug, α-difluoromethylornithine (DFMO) that could decrease cancer invasiveness in vitro and in vivo. After DFMO treatment, the traction force of MCF-7 was decreased, whereas that of MCF-10A was not changed. While various methods have been conventionally used to study the mechanical properties of cell (e.g., AFM, optical tweezers, magnetic tweezers, optical stretcher, and micropipette aspiration), as well as forces that cells exert (e.g., traction force microscopy, and micro-post array), these technics cannot measure intracellular forces associated with alteration of intermolecular interactions. To explore molecular forces involved in cellular responses, recently, new methods such as FRET (Förster resonance energy transfer) based force sensors and DNA based molecular force sensors (hairpin molecular force sensor and DNA sequence based tension gauge molecular tether) have been developed. Förster resonance energy transfer (FRET) utilizes energy transfer between two fluorescent molecules by overlapping excitation and emission wavelength such as CFP-YFP and EGFP-mCherry pairs 72. Thus FRET efficiency is determined by conformational change of fluorescent molecules, e.g., the orientation and distance between two fluorophores. FRET is usually applied for biomolecular activation sensors such as focal adhesion kinase (FAK) 73, Src kinase 74, and the GTPase proteins 75, where FRET sensors are encoded in proteins within cells genetically to measure pN forces directly. Various types of FRETbased force sensors are developed to measure the force associated with structural changes of proteins. F Meng et al. initially developed a molecular force sensor, known as stretch-sensitive FRET (seFRET) whose sensitivity is from 5 to 7 pN 76-77. They showed that tension in α-actinin and filamin was lower at the lagging edge where filopodia were missing than leading edge during cell migration. Furthermore, 8


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they developed spectrin stFRET (ssFRET) using spectrin repeat domains as the linker to investigate intramolecular forces in focal adhesion complexes

77

. C Grashoff group designed a tension sensor

module (TSMod) using a 40-amino-acid sequence as the linker between mTFP1 and Venus fluorescence molecules

78

. They demonstrated that the molecular tension that mediated vinculin assembly was

increased at the protrusion during cell migration. FRET-based molecular force sensors, therefore act as a powerful tool to unveil the role of molecular tension in various protein machineries such as vinculin 79-80

, E-cadherin 81, PECAM-1 82, and integrin 83.

DNA hairpins and tension gauge tether (TGT) are well-known DNA-based molecular force sensors. DNA hairpin force sensor is tagged with one fluorescence dye and a quencher for FRET in single strand DNA with RGD loops 84. When transmembrane protein integrin applies force across RGD ligand, the molecular force sensor emits lights like a switch. In these molecular sensors, G-C content determines the single molecular force when it turns on/off, i.e., 22% G-C for 4.7 pN ± 1.7 pN and 100% G-C for 13.1 ± 2.4 pN. Using this method, the cellular traction force by focal adhesions and tension force between T-cell receptors and ligands were investigated

85-86

. Unlike DNA hairpin sensors, TGT is

composed of double-strand DNA and rupturing force between two strands of DNA is utilized as a force sensing molecular unit 87. TGT sensors can be designed to break at different magnitude of forces ranging from 12 to 56 pN. These forces are determined by a magnetic tweezer. Ha and coworkers have recently demonstrated that a cell needs a thresheold magnitude of single molecular force (40 pN) between integrin and ligand to adhere the cell onto the substrate during initial spreading

87

and such single

molecular force across integrin to adhere on the substrate was not dependent on cell types (CHO-K1 cell, Breast cancer cell HeLa, and MDA-MB-231) 88. To identify diverse biological processes governed by cellular mechanics, a variety of molecular sensors have been developed. Cancer cells generally have larger traction force than the normal cells, and highly metastatic cancer cells can exert stronger force on the substrate than less-metastatic cancer cells. However, cellular mechanical force of cancer cell and non-cancerous cell using aforementioned molecular force sensors has not been reported yet.

4. Cancer mechanosensation The cancer microenvironment can enhance or inhibit cancer cell behaviors such as sensitivity of cellular microenvironment, motility, polarization, cytoskeletal organization, and proliferation via mechanical signal transmitting process termed mechanotransduction. To decipher these mechanotransduction pathways, various bioengineering platforms that manipulate cellular microenvironments have been developed. Four major approaches - ECM stiffness control, micropatterned cell confinement, 9



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micro/nano topographic substrate, and ECM specificity - are introduced as representative methods for microenvironmental mechanosensing of cancer cells. Details of their material properties and biological observations are summarized in Table 2. 4.1 ECM stiffness Cancer cell can actively recognize the physics of extracellular microenvironment through the mechanosensory proteins, integrin adhesion network

89

. With these mechanosensory proteins that

connect ECM to cytoskeleton, signal cascades are involved in interpreting extracellular mechanical signals to biochemical signals. To discover the interaction between cancer cell and ECM stiffness, various substrates with diverse compliant materials instead of traditional tissue culture flask or rigid glass have been used. Polydimethylsiloxane (PDMS) is widely used in diverse research fields due to its high biocompatibility and stiffness-controllable property. Recent studies have shown that migration, invasive ability, and activities of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) of adenoid cystic carcinoma cells (ACC2) and lung cancer cells (A549) are regulated by substrate stiffness via RhoA/ROCK pathway

90-91

. With increasing substrate stiffness (6-135 kPa),

expression level of MMP2, MMP9, MMP14, RhoA, Rac1, ROCK1, and ROCK2 are up-regulated, while those of TIMP1, TIMP2, and TIMP4 are down-regulated 90. With stiffness ranging from 500-2000 kPa, fibroblastic sarcoma cells (SaI/N) show increased migration speed and random migration (i.e., less persistent migration) with increasing substrate stiffness 92. Prauzner-Bechcicki et al. have reported that proliferation and cell spreading area of prostate cancer cells (Du145, PC-3) and melanoma cells (WM115 and WM266-4) on softer substrate (0.75 MPa) were higher than those on stiff substrate (2.92 MPa) since more fibrinogen could be deposited on soft PDMS substrate (0.75 MPa). Morphological parameters of cancer cells were significantly different by substrate rigidity

93

. Dupont et al. have

identified the role of Yes-associated protein (YAP) and PDZ-binding Motif (TAZ) in mechanosensation of substrate compliance by culturing MDA-MB-231 cells on stiff and soft micropillars array 94. They demonstrated that YAP/TAZ mediated mechanical cues instructed by cellular microenvironment. YAP/TAZ localization in the nucleus on stiff substrate requires Rho GTPase activity and actomyosin contraction. Polyacrylamide (PA) hydrogel is extensively used to mimic mechanical rigidity of extracellular microenvironment of in vivo condition because it is easily synthesized and the range of stiffness values could cover various soft tissues in the body95-98. Various studies have observed cancer cell behavior using PA gel with a wide range of substrate stiffness. Schrader et al. have shown that increasing substrate stiffness (1-12 kPa) can promote proliferation of hepatocellular carcinoma cells (Hun7, HepG2) via 10


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mechanotransduction pathway involving protein kinase B (PKB/Akt) and phosphorylation of STAT 3 (signal transducer and activator of transcription 3) 99. Furthermore, chemotherapy-induced apoptosis of hepatocellular carcinoma cells is decreased on stiff substrate due to reduction of poly-ADP-ribose polymerase cleavage 99. Nukuda et al. have also reported that stiff substrate (126 kPa) can enhance the expression of MMP7 compared to soft substrate (2 kPa). They found that YAP, epidermal growth factor receptor (EGFR), α2β1 integrin, and myosin regulatory light chain (MRLC) increased MMP7 expression in T84 colorectal cancer cells 100. Mcgrail et al. have demonstrated that cell adherent index, traction forces, cell spreading, and polarization of phosphorylated myosin light chain (pMLC) of ovarian cancer cell (SKOV3, OVCAR3) are increased on soft substrate (2.83 kPa) compared to those on hard substrate (34.88 kPa). Mesenchymal phenotypes are more dominant on soft substrate than on stiff substrate, indicating that the malignancy of ovarian cancer cells is increased on soft matrix

101

.

However, other groups have shown opposite results. McKenzie et al. have demonstrated that cell spreading, focal adhesion (vinculin, focal adhesion kinase phosphorylation) formation, traction forces, and myosin light chain phosphorylation of epithelial ovarian cancer cells (SKOV3) are increased with increasing substrate stiffness (3-125 kPa) 102. They also demonstrated that hard stiffness could increase YAP translocation into nucleus and metastasis of cancer cells

102

. Cell stiffness is also an important

index to identify the state of cells and cellular modulus can also be changed in response to substrate modulus. Indeed, with increasing substrate stiffness, cellular stiffness of normal hepatic cell (L02) and hepatocellular carcinoma cell (HCCLM3) are increased 103. Collagen, one of the primary constituents in mammalian ECM, is conveniently used to prepare rigiditytunable in vitro cell culture matrix. Increased secretion of collagen around tumor is known to promote invasion and metastasis of cancer cells 104-105. Intracellular compliance of prostate cancer in 3D collagen matrix is increased with increasing collagen matrix (stiffness range: 0.16 to 8.73 Pa) 106. Matrix ligand density increases with increasing collagen matrix stiffness since pore size of collagen matrix is correlated with matrix mechanics and ligand mediated cell-matrix interaction 107. In the same manner, breast epithelial cell (MCF-10A) in stiff 3D collagen matrix shows higher invasion with increased matrix anchorage (β1 integrin) through Src kinase, PI3K and Rac1 108. During this process, expression level of membrane-tethered proteinase (MT1-MMP) encoded by a mesenchymal gene is up-regulated for invasion behavior of cancer cell through small pore size of collagen matrix. Polyvinyl chloride (PVC) based substrate is known to provide better image contrast between cell and the medium likewise conventional culture ware. By mixing carboxylate type plasticizer (DINCH) and PVC, stiffness of PVC is tunable within stiffness range of 20.2 to 61.1 kPa. Filopodia (expanded microspikes around cell edges), are generally observed in highly invasive cancer cells 109. Filopodia of lung adenocarcinoma cells (CL1-5) are more dynamic on soft substrate with higher density and longer 11



filopodia

110

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. Activities of filopodia are modulated by cell-substrate adhesion strength via myosin

contractility. Soft substrate induces dense and long filopodia that can facilitate cancer cell spreading and invasion into ECM or surrounding tissue with more explored directions 110. Matrix stiffness plays an important role in various activities of cancer cells, including progression, proliferation, attachment, mesenchymal phenotype change, migration and invasion. Cancer cells on soft substrate have more dynamic interaction with surrounded microenvironment to search for external cues for migration, whereas cancer cells on stiff substrate are relatively ready to be activated for malignant transformation by up-regulating MMP, β1 integrin, and focal adhesion kinase (FAK). 4.2 Micropatterned cancer cell confinement With recent advance of microtechnology, robust methods have been developed to control the cellular microenvironment to characterize cellular properties. One representative method that has been widely used to confine cells in a specific microenvironment is micro-patterning of cell adhesive proteins or non-adhesive materials on substrate 111-112. Microcontact printing (μCP) technique allows a substrate to be functionalized with a wide range of adhesive proteins at size of nanometers. Fibronectin is one of the most widely used proteins in μCP experiments because it is an extracellular cell-adhesive protein that plays a major role in cell motilities such as initial cell attachment, growth, migration, and wound healing process

113

. Recently, numerous studies have been performed to comprehend cancer cell-

substrate interaction with μCP technique. Bischofs et al. have found that cell shape reorganization is regulated by actin cytoskeletons and activated myosin II motors. They cultured melanoma cells (B16 cells) on microcontact printed fibronectin as a discrete point of cell adhesion. Mature adhesion sites and focal adhesions were developed overlying the microcontact printed fibronectin while actin fibers were connected as an arc-like border at outline of cellular periphery 114. Crossbow-shaped or teardrop-shaped adhesive micropatterns are commonly adopted in single cell confinement since these micropatterns can induce polarized cell shape which is observed in cell migration in nature

114

. In crossbow-shaped

micropatterns, shear modulus of breast cancer cell (MDA-MB-231) is decreased from cell rear to the front along the polarity axis of the micropattern

115

. Interestingly, intracellular distribution of

microtubule networks in cancer cells and normal cells on crossbow-shaped pattern were different. Microtubules of cancer cells (MDA-MB-231) are distributed more homogeneously with similar bundle size while those of nontumorigenic cells (MCF-10A) show bundles in the cell center. Aggarwal et al. have used microcontact printed fibronectin at width of 75 and 150 μm to explore nuclear shape deformation during cell migration of breast cancer cells (MDA-MB-231) and breast epithelial cells (MCF-10A) 116. They found that cells could spread and become fully elongated within 25 min on 75 μm patterned fibronectin. Moreover, during mechanosensation, physical stress transferred from 12


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extracellular microenvironment is transmitted to the nucleus but nuclear shape abnormalities induced by cell shape deformation is detected only in cancer cells 116-118 . Such mechanical signals affected cell and nucleus abnormalities as well as tumor growth 119. For tumor cells on confined area of fibronectin, mechanical stimulation can be directly induced by pressing cells using agarose gel as cushion 11. The phenotype of cells in leading edge is changed to be more invasive when compressive stress is induced. Microcontact printing with self-assembled monolayer on gold coated substrate is also widely used to pattern cells. Moreover, understanding the directional motility and bias motion of cancer cells is important since they are relevant to physiological processes of metastasis. Grzybowski group has fabricated micropatterned ratchets to facilitate directional cell migration

120

. Interestingly, in specifically designed micropattern,

where linear ratchets with spikes inclined at 45° (short base of a unit trapezium), two types of cancer cells (B16 cell and MDA-MB-231) moved in opposite directions. Cell-cell communication between cancer cell and stromal cell or between cancer cell and other tissue cells plays an important role in cancer development. To speculate how stromal cells affect cancer progression, more than two types of cells can be cultured together. Co-culture system with precisely controlled microenvironment is often used to understand how cancer cells affect properties of noncancerous cells, and vice versa, either directly affect them through physical/chemical cell-cell interactions or indirectly through soluble cytokines. To mimic these co-culture systems, cell micropatterning using μ-eraser technique 121 or stencil micropatterning technique 122 has been developed. Zhong et al. have cultured lung cancer cells (A549) on poly(lactide-co-glycolide) (PLGA) and then removed A549 cells on specific region by pressing cell layer with PDMS stamp 123. After using μ-eraser technique, osteoblast cells (hFOB 1.19) were seeded to investigate the effect of co-culture on two cell types. Such method is also used for drug evaluation since bone is one of the main organs metastasized by lung cancer 123. The efficacy of doxorubicin was decreased whereas alkaline phosphatase expression of osteoblast cells was elevated in the co-culture system. Shen et al. have co-cultured breast cancer cells (MDA-MB-231, MCF-7) with relevant stromal cells, e.g., breast fibroblast, cancer-associated fibroblasts and bone marrow-derived mesenchymal stromal cells (BMSC), using micro-patterned tumor-stromal assay 124. Among them, co-culture of MCF-7 and BMSC showed the highest increase of MMP14 expression. When cancer-stromal interaction inhibitor, e.g., reversine was used to treat the coculture model and in vivo model, cancer growth and metastasis were suppressed by reducing of stromal cell activation 123. 4.3 Micro/nano topographic substrate Topographic features of cancer cell microenvironment can also change cell motility and cancer 13



Page 14 of 46

progression. For instance, cancer cells can recognize the collagen fibers and migrate along with the same direction of collagen fiber

125-127

. Generally, the ECM recognition, mechanotransduction, or

migration pathway of cancer cells is not different from that of normal cell, i.e., non-cancerous cells 128129

. However, cancer cells have higher elasticity than normal cells and thus are highly invasive as

mentioned earlier. Migration and invasion of cancer cells are complicated processes. They are affected by a combination of external signals. To explore motility mechanism underlying cancer invasion, recent studies have determined how topographical stimulations could affect cancer behavior by culturing cancer cells on micro/nano topographic substrate. Fibroblastic sarcoma cells (SaI/N) placed on dense microcircular posts or microgrooved pattern showed increased persistence and migration speed compared to those lying on flat substrate, indicating topographic variation guided cells to migrate less randomly 130. Kushiro et al. have compared migration behaviors of cancerous cells and non-cancerous cells using micro-fabricated substrate with PDMS microgrooved wall 131 and tetra-(polyethylene glycol) (tetra-EPG) microgrooved wall

132

. Non-cancerous breast epithelial cells (MCF-10A) and prostate

epithelial cells (RWPE1) migrated along the microgrooved wall with increased migration speed and persistence length whereas cancerous breast epithelial cells (MCF-7) and prostate epithelial cell types (RWPE2, PC-3) exhibited less cell-substrate interactions, such as less persistent migration, climbing microgrooved walls with less aligned cytoskeleton. These cancer cell behaviors suggest that cancerous cells may ignore geometrical cues and migrate more randomly to increase cancer malignancy. Comparisons of non-cancerous breast epithelial cells (MCF-10A) and breast cancer cells (MDA-MB231, MCF-7) have also been performed under the topographic cues 15. While the proliferation of cancer cells was not changed regardless of substrate topography, that of non-cancerous cells was significantly decreased when cells were cultured on 2-4 μm microgrooves or micropillars substrates. In the same manner, cancer cells were resistant to Rho-ROCK-Myosin signaling activated by topographic cues. They consequently continued uncontrolled proliferation 15. 4.4 ECM specificity Extracellular matrix (ECM) is a collection of extracellular macromolecules secreted by various types of cells and acts as a key component of tissue. It provides the physical and biochemical support of an organ. ECM has a wide range of mechanical and various biochemical properties. ECM can change shape, polarization, proliferation, apoptosis, motility, and development of cancer cells via mechanotransduction signals of cell-ECM. ECM also plays a role as a blocking barrier to prevent drug diffusion to cancer cells, causing chemoresistance or inhibiting cancer cell migration and invasion to neighboring tissues. Many studies have investigated the relationship between cancer cell behaviors and specific ECM. Zhang et al. have compared properties of breast cancer cells (e.g., SUM159, MDA-MB14


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468) cultured on various ECM-coated PDMS to alter their physicochemical properties 133. Cancer cells were cultured as a monolayer on polystyrene, collagen-, fibronectin-coated PDMS substrate. However, SUM159 cells exhibited a round shape instead of a spindle shape on BSA coated PDMS, where the round shape was a representative morphology of cancer stem cell. BSA coated PDMS promoted cancer stem cell population by down-regulating pSTAT3 and pAkt proteins 133. While BSA is not a component of ECM, it is one of the common surface-modifying molecules to increase wettability. However, cell adhesion experiment with BSA coated substrate could lead to incorrect results since BSA is a highly soluble protein. Thus dissolved albumin can interact with numerous ligands in the cell as well as bioactive factors that could affect cell fate, which can lead misinterpretation of their results 134-135. Laminin-332 (also known as laminin-5), an ECM component with α3Aβ3γ2 chain composition, is secreted by cancer and stromal cells and play a critical role in epithelial cell adhesion to the basement membrane 136. Some studies have reported that expression of laminin-332 is increased in several types of cells such as gastric carcinoma, oral squamous cell carcinoma, and hepatocellular carcinoma cells 137-139

. Laminin-332 binds to β4 subunit of integrin and promotes migration, invasion, and proliferation

of tumor cells. Furthermore, transforming growth factor-β and laminin-332 are involved in epithelialmesenchymal transition (EMT) 140-141. In cancer-stromal interface, laminin-332 and membrane type 1MMP are overexpressed, consequently promoting tumor invasion by remodeling microenvironment 142. Periostin (also known as osteoblast-specific factor 2, POSTN, PN) is an ECM protein secreted by connective tissues such as tendons, myocardium, skin, and bone. It interacts with diverse integrins, e.g., αVβ3, αVβ5, and α6β4. Integrin-bound periostin activates various cancer cell behaviors including EMT, migration, invasion, and proliferation 143-145. In addition, periostin is known to be a key component of cancer stem cells-supportive niches by increasing Wnt signaling 146. Fibronectin plays an important role in cell adhesion by binding integrin at the cell membrane. Fibronectin-binding cells exert force through integrin family such as α3β1, α4β1, α5β1, αVβ1, αVβ3 and αVβ6

147

. A wide range of cell types can bind to fibronectin with these receptors, consequently

affecting various cancer cell behaviors 148. Among these integrins, α5β1 integrin is markedly active in malignant cancerous cells

149

. Generation of fibronectin fibril is essential to physiological and

pathological processes such as actin-myosin contraction, cell adhesion, development, invasion, and differentiation

150-151

. Park et al. have found that exogenous fibronectin can promote EMT by up-

regulating cadherin, vimentin, Snail, and MMP2 152. They also reported that fibronectin-induced EMT was dependent on Src kinase, ERK/MAP kinase, and transforming growth factor (TGFβ). Hyaluronan (also known as hyaluronic acid, HA) is a macromolecular polysaccharide found in ECM. It is a major component of glycosaminoglycan (GAG). HA expression is increased in various cancers such as breast, prostate, bladder, stomach, and pleura cancers 153. HA synthases (HAS) such as HAS1, 15



Page 16 of 46

HAS2, HAS3 play an essential role in all stages of cancer metastatic process

154

. On the contrary,

degeneration or degradation of HA by bovine hyaluronidases or PEGylated human recombinant PH20 hyaluronidase can induce promising tumor growth inhibition while increasing chemotherapeutic effects 153

.

5. Metastasis mechanics Metastatic dissemination, spreading of cancer cells from primary or initial site to surrounding or distant sites, is a unique characteristic of cancer cells, which typically involves a series of sequential processes of EMT, invasion, intravasation, and extravasation. In order to initiate and complete the metastatic sequential, cancer cells must change their shape to invasive phenotype (EMT), invade to surrounding cells or ECM (invasion), penetrate the circulatory system (intravasation), and exit from blood or lymphatic microvessel (extravasation) 146, 155-156. Cancer metastasis has been studied for more than one century. One famous hypothesis is “seed and soil” proposed by Stephen Paget in 1889. It means that cancer metastasis depends on cross-talk between cancer and the specific microenvironment of organ. This hypothesis has been widely accepted and provides a guide to cancer and metastasis research 155, 157. Supporting the hypothesis, recent advances in micromanipulation and imaging technology have unveiled the importance of mechanical signals in metastatic processes.

11, 158-159

. This chapter will

recapitulate the crucial role of mechanotransduction feedback loop in metastasis, interplaying between biophysical stimuli and biochemical responses. 5.1 EMT EMT can alter cellular properties from epithelial phenotype, high adhesiveness, and low migratory ability to mesenchymal-like phenotype with neoplastic property, low adhesiveness via loss of Ecadherin, and increased invasiveness. Mechanical stimuli generated by cellular microenvironment stiffness can affect the regulation of EMT by TGFβ, a potential EMT inducer 160. In addition, EMT can be regulated by mechanotransduction pathways through transcription factors, twist-related protein1 (TWIST1)

161

. Nuclear translocation of TWIST1 is promoted by releasing TWIST1-binding proteins,

Ras GTPase-activating protein-binding protein 2 (G3BP2) in stiff matrix. Many proteins are known to characterize EMT such as E-cadherin, vimentin, YAP, and transcriptional co-activator TAZ 162-163. Loss of E-cadherin, increase of vimentin, and nuclear localization of YAP have been observed in EMT process. Elevation of specific ECM including laminin-332, periostin, and fibronectin has been observed in EMT as mentioned above. Although these phenomena with 3D in vitro studies 164-166 provide robust mechanistic insights to EMT of cancer cell, promising results are not simply derived or matched with 16


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in vivo models 167-168. 5.2 Cancer cell invasion To penetrate the circulatory system (intravasation process), cancer cells start to migrate and breach an extracellular basement membrane (BM), a sheet-like specialized ECM that acts as natural barrier of primary tumor. The BM is composed of reticular connective tissue and basal lamina containing multiple molecules of type IV collagen, laminin, entactin/nodigen, and proteoglycans of BM invasion by cancer cell has been extensively reviewed previously

169

. Detailed mechanism

170-173

and various in vitro

assays have been adopted to elucidate the underlying mechanism of cancer cell invasion 174-176. Increase of tumor stiffness with type I collagen and fibronectin enrichment can lead to cancer cell proliferation and invasion 16, 177-178. Invasive cells feature invadopodia, distinct actin-based dynamic protrusions that can degrade ECM component by recruiting MMPs

179

. These extended membrane protrusions are

enriched by invasion-related complex such as cortactin, paxillin and protein kinase C

180

invasion is enhanced in response to mechanical stimulation with increased invadopodia

. Cancer cell

181

. Applying

transient tugging forces to collagen and fibronectin substrate can down-regulate β1 integrin, Rac1-GTP, PAK1 activation, LIM kinase 1 activation and up-regulate cofilin activation with longer and matured invadopodia. Recent study has reported that colorectal carcinoma cell sphere can disseminate to peritoneum without EMT process

182

. They showed that tumor sphere can maintain epithelial

organization to enrich E-cadherin and β-catenin between cancer cells. In addition, tumor sphere is polarized to the outward of sphere that can be observed by apical markers including ezrin, villin, atypical protein kinase C, CD133, phospho-ezrin-radixin-moesin, and Na+/H+ exchanger regulatory factor. Furthermore, tumor sphere can maintain its spheroidal shape and collectively migrate into collagen gel or matrigel. Whether EMT is an essential process for invasion and metastasis or not is not clear that the correlation between EMT and metastasis will be an exciting area for future research 182. 5.3 Intravasation Following metastatic cascade of EMT process and local invasion, cancer cells will enter into the blood or lymphatic circulatory system (intravasation) for spreading nearby or at distant sites. Intravasation leads to circulating tumor cells (CTCs) in circulatory system. These CTCs present high risk for secondary metastasis. Cancer cell intravasation depends on penetration through connective tissue and cell-cell junction between endothelial cells. In this process, the shape of cytoplasm and nucleus can be severely deformed unless endothelial cells and cell junction are retained. Molecular mechanisms of intravasation have been recently reviewed

183

. A number of studies have also discussed cancer

intravasation using in vitro and in vivo 3D modeling. 17



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In vitro systems including transwell invasion assay and artificial micro-vessel system using microfluidic device have been developed to elucidate or visualize cancer intravasation. Kamm group has developed microfluidic chip-based platform to mimic tumor-vascular interface in 3D 184. Invasion into endothelial barrier of fibrosarcoma cells (HT-1080) was visualized in the presence of tumor necrosis factor alpha. They have also developed 3D microfluidic model of breast cancer cell metastasis to bone that mimics osteo-cell conditioned microenvironment with vascular system. Tri-culture compositions of three different cell types (endothelial cells, breast cancer cells, and osteogenic differentiated cells) from bone marrow-derived mesenchymal stem cells were also established to recreate bone specific metastasis of breast cancer cells. Intravasation of cancer cells is related to RhoA GTPase signaling 185. Activation of RhoA stimulated by contacted macrophage can trigger actin-based dynamic protrusions termed invadopodia. For in vivo intravasation studies, two methods are commonly utilized. One is quantitative measurement of intravasation by analyzing circulating tumor cells, while the other is obtaining intravital imaging of cancer cell invasion through blood vessel wall. Segall group has reported that overexpression of epidermal growth factor receptor in adenocarcinoma cells (MTLn3) can enhance intravasation and metastasis to lung

186

. Direct imaging of cancer cell intravasation was conducted using multiphoton

microscopy 187. Advanced in vitro microenvironments with increased complexity and in vivo intravital imaging will promote understanding of the interaction between cancer cells and stroma during metastasis. 5.4 Extravasation For complete metastasis, cancer cells in blood or lymphatic circulatory system (called CTCs) have to adhere to the capillary and exit to form metastasized tumor at secondary site. The first step for extravasation of cancer cells survived from shear stress in the circulatory system is attachment to endothelial cells. Endothelial cell adhesion is then needed to stabilize cancer cells that will exit the circulatory system by squeezing the cytoplasm and nucleus. A number of extravasation and intravasation studies have been performed using in vitro and in vivo 3D modeling 188-191. Extravasation can be classified as paracellular extravasation and transcellular extravasation based on route of cancer cell extravasation. Paracellular extravasation is related to adhesion molecules (including E-selectin, Pselectin glycoprotein ligand 1, CD24, CD44, mucin 1, and galectin-3-bining protein) and chemokines and their receptors including CXC-chemokine ligand 12, CXC chemokine receptor type 4 (CXCR4), and CXCR7 192. Transcellular extravasation is recently observed in in vitro study using colorectal cancer cells and leukocytes

193-194

. With this mechanism, cancer cells can exit the circulatory system and

penetrate through endothelial cells, not between endothelial cells. However, further studies will 18


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determine whether this extravasation exists for other type of cancer cells or in vivo.

6. Molecular mechanism of cancer mechanosensation Physical microenvironments and mechanical stimuli can regulate properties of cancer cells, including proliferation, cytoskeleton distribution, migration, gene expression, and signal transduction. These abilities are known as cancer mechanosensation that occurs at the interface of cancer cell/ECM or cancer cell/stromal cell, mainly through integrin of cancer cell and integrin ligand of ECM such as fibronectin, collagen, elastin, and laminin. Integrin mediated cell membrane and focal adhesion complexes such as paxillin, FAK, and Src kinase are activated by cancer cell-ECM adhesions. These focal adhesion complexes can interact with actomyosin cytoskeleton to transmit mechanical signal to the nucleus from ECM. Mechanical inputs such as rigidity of ECM, grooved or patterned surface can regulate mechanoinduced transcriptional activator, YAP/TAZ to induce proliferation, survival, metastasis, and cancer stem cell attributes. EMC stiffness, cell density, and cytoskeletal tension are known to regulate expression of YAP and TAZ 94, 100, 195-196. YAP and TAZ of cancer cells are localized in the nucleus when cells have high cytoskeletal tension and when cells are cultured on stiff substrate. However, they are relocalized in the cytoplasm when cells are cultured on small confined area or soft substrate. Regulation of YAP and TAZ activation depends on small GTPase Rho-ROCK-myosin signaling pathway 90-91, 197. The activation of Rho-ROCK-myosin signaling pathway is regulated by β1 class integrins such as α5β1 198

. Since activations of YAP and TAZ are hallmarks of cancer development, specific targeting YAP and

TAZ might be an effective strategy for cancer therapy 199. One of the well-known regulators of cancer development is TGFβ which is cytokine of transforming growth factor superfamily that includes TGFβ1 to 4, activin, and inhibin 200. TGFβ is a key regulator in metazoan biology. It regulates cell division, cell growth, death, survival, and EMT

201

. Activation of

TGFβ is also dependent on the condition of ECM. The rigidity of ECM can change the response of TGFβ activation through phosphatidylinositol 3-phosphate/Akt signaling pathway 202-203. Stiff substrate lowers the threshold of TGFβ activation. The activation of TGFβ will trigger phosphorylation of signal transducer, SMADs. TGFβ activation also induces intracellular translocation of SMADs from the cytoplasm into the nucleus

202

. Receptors of TGFβ can phosphorylate SMAD2 and SMAD3. SMADs

are localized at the nucleus when TFGβ is activated

201, 204

. TGFβ signaling pathway plays a role as a

tumor suppressor in early cancer development such as growth inhibition and apoptosis induction. In contrast, cancer progresses such as EMT, migration, invasion, metastasis are promoted by this pathway. Therefore, targeting TGFβ as a cancer treatment is a challenging strategy to develop therapeutics 205-207. Wnt signaling is also an important cascade that regulates cancer development. Wnt/β-catenin signaling 19



Page 20 of 46

pathway controls cancer behaviors such as migration, proliferation, and survival. In canonical signaling pathway, β-catenin, a subunit of cadherin, acts as signal transducer. It is accumulated in the cytoplasm and protected by Adenomatous polyposis coli (APC) when Wnt signaling is inactivated. β-catenin is translocated to nucleus where it acts as a transcriptional co-activator 208-209. Together with YAP and TAZ, β-catenin is also localized in the nucleus when cells are cultured on stiff ECM 210. Ovarian cancer cells cultured on stiff matrix show β-catenin localization in the nucleus with down-regulation of Wnt signaling inhibitor, dickkopf-1

211

. In addition, β-catenin localization in the nucleus is observed when

hepatocellular carcinoma cells are cultured on stiff ECM with elevated phosphorylation of glycogen synthase kinase 3β, GSK3β

212

. To inhibit cancer cell proliferation and metastasis, inhibition or

activation of specific proteins such as porcupine, vacuolar ATPase, tankyrase, PP2A, and GSK3β involved in Wnt signaling can be conducted. The complexity of the Wnt/β-catenin signaling pathway provides putative cancer therapies without causing side effects 213-215.

7. Concluding remarks Cancer mechanobiology is an emerging field of medical biology that can unveil the underlying mechanisms of cellular mechanosensation and cancer metastasis. Advances in mechanobiology and microtechnology provide robust prospect to discover links between mechanical stimuli and cellular responses such as cell stiffness, migration, polarization, death or survival, cytoskeletal reorganization, and relocation of subcellular organelles. In this review, we summarized various methods used to measure cancer cell modulus (e.g., AFM, optical/ magnetic tweezers, and micropipette aspiration) and cancer cell-substrate interaction forces (e.g., traction force microscopy, micro-post array, FRET, TGT, and hairpin DNA). The modulus of cells is changed depending on the level of cancer transformation and diverse cell types, although the well-known truth is that most cancer cells are softer than normal cells. Accordingly, we also summarized that cell stiffness, migration speed, MMPs, FAK, and β1 class integrins were up-regulated in stiff ECM, whereas invasiveness, length and density of filopodia were up-regulated in soft ECM. In addition, metastasis mechanics were introduced depending on stages of metastasis including EMT, invasion, intravasation, and extravasation. Although there is compelling indication for mesenchymal phenotype in initial stage of metastasis in vitro as well as in vivo experiments, recent study has reported invasion of colorectal carcinoma cell sphere to peritoneum without EMT process. Understanding and targeting all metastasis stages of various cells will provide promising effective cancer therapies. Finally, the molecular mechanisms of cancer mechanosensation that promote the cancer metastasis were discussed in terms of integrins, TGFβ, and Wnt/β-catenin signaling pathways. These can be used as therapeutic targets. Toward this end, improvement of 20


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mechanobiology with cancer research will provide more effective cancer therapies by regulating cancer microenvironment and transcriptional factors.

8. Acknowledgements Authors thank Dr. Dong-Hwee Kim’s Applied Mechanobiology Group (AMG) at Korea University and Dr. Hae-Won Kim’s Institute of Tissue Regeneration Engineering (ITREN) at Dankook University for thoughtful discussion. This work was supported by the KU-KIST Graduate School of Converging Science and Technology Program, Korea University Future Research Grants, and National Research Foundation of Korea (2016R1C1B2015018).

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Krishnamurthy, N.; Kurzrock, R., Targeting the Wnt/beta-catenin pathway in cancer: Update

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FIGURES

Figure 1. Representative experimental tools for measuring physical properties of the cell. (a) Atomic microscopy, (b) Optical tweezer, (c) Magnetic tweezer, (d) Optical stretcher, (e) Micropipette aspiration, (f) Microfluidic assay, (g) Microrheology

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Figure 2. Measurement and principle of intracellular forces. (a) Traction force microscopy: movement of micro beads embedded in hydrogel substrate, (b) Micro-post array: magnitude of post deformation, (c) FRET tension sensor (TSMod): FRET efficiency between donor (mTFP) and acceptor (venus) fluorescence proteins linked by amino-acid chain, (d) DNA based force sensor (hairpin molecular force sensor): switch the fluorescence signal as exerting cellular force, (d) DNA based force sensor: DNA sequence based tension gauge molecular tether.

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Figure 3. Schematic of cancer metastasis. Aggregation of primary cancer cell grows out by the ECM proteins at initial stage of metastasis, when some cancerous cells follow epithelial to mesenchymal transition with EMT-associated features, including loss of E-cadherin and increase of N-cadherin, vinculin, and nucleus translocation of YAP/TAZ, TWIST1, G3BP2. Before penetrating the circulatory system, cancer cell breaches basement membrane (BM) at invasion stage. In this stage, MMPs recruiting, invadopodia, integrins and FAs are activated. Moving to distal sites, cancer cells start to penetrate circulatory system with intravasation features such as lose cell-cell junction, RhoA activation, increased TGFβ, and overexpression of EGF receptors. Once, a cancer cell penetrates into the circulatory system, CTC floats with the blood stream and then exits from circulatory system to form metastasized tumor at secondary site in response to environmental cues.

42


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Figure 4. Molecular mechanism of cancer cell mechanosensation. Cancer cells respond to mechano stimuli such as ECM stiffness, micro/nano topography and cell confinement. Mechanical signals are transmitted to focal adhesions such as paxillin, FAK and Src through integrin that activate small GTPase Rho, ROCK and myosin in series. In addition, mechanical signals such as matrix rigidity induce activation of TGFβ and Wnt/β-catenin. These activations lead nuclear translocation of transcription factors such as YAP, TAZ, SMAD and β-catenin. Mechanosensation of cancer cell promotes cell proliferation, survival, and MMP secretion that are required for cancer metastasis.

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Table 1. Physical properties of the cancer and normal cells Mechanical cue

Cell source

AFM

Breast

Ovarian

Prostatic

Bladder

Breast Tissue (Patient)

Optical tweezer

Micropipette aspiration

Vulvar Tissue (Patient) Endometrium Tissue (Patient) Breast

Colon Prostate

Microfluidic cytometry Optical stretcher

Breast

Breast

Cell line MCF-10A (non-malignant epithelial cancer cell) MCF-7 (malignant epithelial cancer cell) A184A1 (normal) T47D (cancer) MCF-7 (malignant epithelial cancer cell) HBL-100 (normal myoepithelial cell) MCF-7 (malignant epithelial cancer cell) MDA-MB-231 (basal breast cancer) MOSE (mouse ovarian surface epithelial) Early stage MOSE - Intermediate stage MOSE - Late stage IOSE (non-malignant epithelial) HEY (ovarian cancer) HEY A8 (more aggressive than HEY) PZHPV-7 (non-tumorigenic) PC-3 (adenocarcinoma) Du 145 (metastatic carcinoma from brain) LNCaP (metastatic carcinoma from lymph node) Hu609 (non-malignant ureter) HCV29 (non-malignant bladder urothelium) Hu456 (bladder carcinoma) T24 (bladder carcinoma) Breast tissue (nonneoplastic) Breast tissue (infiltrating ductal carcinoma) Vulvar tissue (nonneoplastic) Vulvar tissue (cancer) Endometrium tissue (nonneoplastic) Endometrium tissue (uterine corpus carcinoma) HBL-100 (normal myoepithelial cell) MCF-7 (malignant epithelial cancer cell) MDA-MB-231 (basal breast cancer) HBL-100 (Myoepithelial) MDA-MB-231 (basal breast cancer) SW48 (high-grade colon cancer) HT29 (low-grade colon cancer) PC-3 (adenocarcinoma) PrEC LH (prostate epithelial) MCF-7 (malignant epithelial cancer cell) MDA-MB-231 (basal breast cancer) HL-60 (human promyelocytic leukemia) MCF-10A (non-malignant epithelial cancer cell) MCF-7 (malignant epithelial cancer cell) MDA-MB-231 (basal breast cancer)

Elasticity / Deformability

Ref

0.5 ~ 1 kPa

26

0.4 ~ 0.6 kPa 2.26 ± 0.56 kPa 1.20 ± 0.28 kPa 1.24 ± 0.46 kPa 95.4 ± 43.6 kPa 87.3 ± 47.8 kPa 55.6 ± 20.1 kPa 1.097 ± 0.682 kPa 0.796 ± 0.549 ± 2.472 ± 0.884 ± 0.494 ± 3.09 ± 1.95 ± 1.36 ±

0.441 kPa 0.281 kPa 2.048 kPa 0.529 kPa 0.222 kPa 0.84 kPa 0.47 kPa 0.42 kPa

28

27

31

32

28

0.45 ± 0.21 kPa 12.9 ± 4.8 kPa 10.0 ± 4.6 kPa 0.4 ± 0.3 kPa 1.0 ± 0.5 kPa 1.16 ± 0.20 kPa 0.47 ± 0.15 kPa / 1.54 ± 0.17 kPa 3.07 ± 1.60 kPa 1.58 ± 0.85 kPa 1.27 ± 0.13 kPa

33

28

28

28

0.57 ± 0.19 kPa 23.5 ± 10.6 Pa 30. 2 ± 15.0 Pa 12.6 ± 6.1 Pa 27.75 ± 12.71 Pa 8.22 ± 4.69 Pa 56.44 Pa 82.31 Pa 19.95 ± 6.77 Pa 47.72 ± 25.7 Pa 2.4 ± 0.2 kPa 0.97 ± 0.50 kPa 0.53 ± 0.04 kPa

27

10.5 ± 0.8 %

39

35

40

41

43

21.4 ± 1.1 % 33.7 ± 1.4 %

44


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Table 2. Cancer cells mechanosensation Approaches

Materials & Methods

500-2000 kPa

Fibrosarcoma (SaI/N)

130

0.75-2.92 MPa

Prostate cancer (Du145 and PC-3), melanoma (WM115 and WM266-4) cells

216

Hepatocellular carcinoma (Hun7, HepG2)

99

Ovarian cancer cell (SKOV3, OVCAR3)

101

Ovarian cancer cell (SKOV3)

217

0.54-33.7 kPa

Hepatocellular carcinoma cell (HCCLM3)

103

0.16-8.73 Pa

Prostate cancer cell (PC-3)

106

175-515 Pa

Breast epithelial cell (MCF-10A)

108

20.2-61.1 kPa

Lung adenocarcinoma cell (CL1-5)

110

Mirodot array

Melanoma cell (B16)

114

Crossbowshaped Line (10μm)

Breast cancer cell (MDA-MB-231)

115

Breast cancer cell (MDA-MB-231)

116

Breast cancer cell (MCF-7, MDA-MB-231)

11

1-12 kPa

ECM stiffness control

2.83, 34.88 kPa

PA gel

3-125 kPa

Collagen PVC

Fibronectin

Square shape Micropatterned cancer cell confinement

Melanoma cell (B16), Breast cancer cell (MDA-MB-231)

120

Pill off

Oral squamous cell carcinoma (OSCC3), prostate carinoma (DU145)

218

Lung cancer cell (A549), osteoblast cell (hFOB 1) Breast cancer cell (MDA-MB-231)

123

Fibrosarcoma (SaI/N)

130

Microgroove

Prostate cancer cells (PC-3, RWPE2), breast cancer cell (MCF-7)

131

Microgroove, micropillar

Breast cancer cell (MCF-7, MDA-MB-231)

15

Prostate cancer cell (PC-3)

132

Breast cancer cell (SUM159)

133

Breast cancer tissues from patients

142

Lung cancer cell from patients

219

Fibronectin

Breast epithelial cell (MCF-10A)

152

Hyaluronan

Pancreatic ductal adenocarcinoma (PDA)

153

μ-eraser PDMS

Pill off Pillar, line

PDMS

PEG Microgroove hydrogel BSA, collagen, fibronectin ECM specificity

91

Ratchet

Fibronectin, laminin Parylene

Micro/nano topographic substrate

Reference 90

6-135 kPa PDMS

Cell type Salivary adenoid cystic carcinoma cell (ACC2) Lung cancer cell (A549)

6-135 kPa

Laminin-332 Periostin

124

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Page 46 of 46

For Table of Contents Use Only

Cancer Mechanobiology: Microenvironmental Sensing and Metastasis

GeonHui Lee1,*, Seong-Beom Han1,*, Jung-Hwan Lee2,3, Hae-Won Kim2,3,4, and DongHwee Kim1,ǂ

1

KU-KIST Graduate School of Converging Science and Technology, Korea University,

Seoul, 02841, South Korea 2

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan


Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan


Department of Nanobiomedical Science & BK21 PLUS Global Research Center for

Regenerative Medicine, Dankook University, Cheonan 31116, South Korea

46


Cancer Mechanobiology: Microenvironmental Sensing and Metastasis

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