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Cancer is one of the most serious threat to human life. According to the American Cancer Society, in 2012 cancer is expected to cause about 550,000 death in USA, which is equal to more than 1,500 people a day and nearly 25% of death. Up to date, treatments have been clinically applied, including surgery, chemotherapy, radiation therapy, and immunotherapy. The choice of therapy is determined by the origin and grade of the tumor, the stage of the disease, and the status of the patient. However, even though 'cancer war' has been declared for decades, cancer is still one of the diseases with only incremental improvements obtained, which makes cancer the major cause of death. Thus, better understanding that can lead to strategic treatment of cancers is in desperate needs.
Chemotherapy seeks solutions to completely remove cancer cells without damage to the rest of the normal tissue. The development of anticancer drugs can be roughly classified into two main categories, which targets to either essential or non-essential functions for cell survival . The essential functions include cell division in the duplication of DNA, separation of newly formed chromosomes, as well as physiological processes such as microtubule polymerization (for example, taxol) or metabolite synthesis (for example, methotrexate). These cytotoxic approaches intentionally target to all rapidly growing cells and are not specific to cancer cells, although the specificity may be reached at some degree because many cancer cells cannot repair DNA damage but normal cells generally can. By contrast, various non-essential targets have been brought onto the scene. Small-molecule drugs were used to inhibit signaling pathways which might not be essential in normal cells. Thus, it yields a differential effect between normal and cancerous cells. Prominent examples are the tyrosine kinase inhibitors imatinib and gefitinib [2-6]. Imatinib inhibits Ableson cytoplasmic tyrosine kinase (ABL), which is crucial in chronic myeloid leukaemia by a chromosomal translocation, but is not required in normal cells. In addition, success was gained by inhibition of non-essential proteins in adult tissues, such as kinases, epidermal growth factor receptor (EGFR), or HER2/neu [7, 8], which lie upstream in signaling networks, or CD-20 in a variety of B-cell malignancies [9, 10].
2.a.2 Role of mechanics in cancer development
Although chemotherapy has brought in some success, the efficacy remains limited in the later stage of cancer development . Traditionally, cancer is thought as a disease with a group of abnormal cells that exhibit uncontrolled growth, and all abnormal cells should be equally tumorigenic. However, pathologists have classified tumors into many histological subtypes . They showed that, in addition to cancer cells, tumors consist of glandular, nervous, cartilage, bone, muscle, and fat tissue. Clearly, this development of tumor is much more complex that just uncontrolled proliferation; rather, it should consist of a serial of events, involving differentiation, disorganization of tissue pattern, invasion, and recruitment of other cell types at different stage. Thus, in addition to the abnormality of individual cancer cells, it suggests that the context of natural tissue formation could be a better way to think of tumorigenesis.
While most of research focus on cellular responses to the chemical stimuli, such as growth factor, cytokines  and metabolites [14, 15], it should be noted that biological systems are not just governed by chemical factors. In addition to the biochemical/soluble cues, emerging evidences have showed that cellular response to mechanical cues in their microenvironment also plays a role. Cells reflect the change of matrix tension or stiffness by actively adjusting its cytoskeletal organization  and adhesion affinity . For example, in the development of normal tissue architecture, the maintenance of epithelial-mesenchymal interaction is coordinated via the balanced force between the extracellular matrix (ECM) and the cytoskeleton within the adjacent cells. Alternation of ECM may results in increased tension and thereby causing localized growth and tissue morphogenesis . Also, there are increased studies revealing the mechanical signal in endothelial function [19, 20], tissue structure [21, 22], and development [23, 24]. Given that cancer should be viewed as the context of tissue formation , the mechanical signal in cancer development should be considered as important as the chemical cues in the entire process. Usually, the development of anticancer drug begins from cell culture, where the tested cancerous cells were enzymatically isolated and plated in vitro. Since the mechanical context introduced from their native environment has been lost in conventional culture, this approach omits the influence such as force interaction and the mechanical property essential for restoring the original cell behavior, presumably causing the incompatibility and drug resistance often encountered in clinical practices. As such, the development of an in vitro model system which allows the implementation of mechanical stimuli for necessary mechanotransduction becomes more and more important for new strategies in anticancer therapy.
2.a.3 Traditional cell mechanics
Biotechnology has been actively contributing to the implementation of mechanical signaling. Traditional mechanical force include shear stress by fluidic flow , mechanical stretch , and mechanical compression to the entire culture [27, 28]. Such 'directional' or 'displacement-based' application can induce either the orientation of cell polarity  or increase of cell migration and invasion [27, 28]. At material perspective, substrate stiffness could be the mostly widely used parameter to studying the environmental mechanics [29-31]. It is because, considering cells as a deformable substance with tensile pre-stress, attachment to the substrate that is more compliable (softer) will result in the contraction of cell body, therefore changing its mechanical condition. Reports showed that cells migrate faster in softer gel and optimize the proliferation in a specific range of stiffness . Stiffness also affects the differentiation and morphogenesis. When myoblasts were cultured on collagen strips on varied elasticity, it shows that myosin/actin striations emerge only with stiffness typical of normal muscle . More interestingly, based on this finding, with a wider testing range of stiffness, mesenchymal stems cells showed multi-lineage differentiation as a function of substrate stiffness; stem cells differentiate into neural cells on softer gel (0.1-1 kPa), which is as soft as normal neural tissue, but differentiate into osteoblasts on harder gel (25-40 kPa), which has similar hardness as normal bone tissue . Together, those studies demonstrate how the change of mechanical cues is seamlessly integrated with biological functions essential for cell/tissue organizational process.
2.a.4 Microtechnology for cellular mechanics
As mentioned above, numerous studies have been done to apply the mechanical stimuli that drive the mechanotransduction signal cascade. However, most of the works are using macroscale forces to the entire tissue culture. Thus, it is hard to dissect the mechanical factor when multiple possibilities are coupled together. For example, using the displacement-based force, i.e., stretch or compression, it is difficult to distinguish the contribution from either the 'quantity' or the 'gradient' of stress along the applied direction. Also, changes of substrate stiffness by adjusting the chemical composition of gel may results in the variation of cell-adherent ligand as well as the material porosity, surface chemistry, and strength of cross-linking, therefore posing the uncertainty to differentiate these matrix properties to the stiffness.
In recent decades, cell microtechnology has been demonstrated as a powerful tool to investigate cellular biology [29, 32-37]. In contrast to the macroscale manipulation, microtechnology has offered the geometric advantages that provide spatial cues in the scale similar to the size of cells. For example, using plasma lithography to create a pattern for cell attachment, researchers had shown that intercellular alignment propagates for a long distance from a geometric edge, potentially explaining the muscle tissue alignment in the natural development . In addition, cell micropatterning itself is capable to stimulate mechanical stress. Cell proliferation can be triggered by a gradient of mechanical stresses generated within the multicellular sheet on a cell-adherent island, therefore setting out to ask whether the spatial arrangement of a population of cells could initiate patterns of spatial asymmetries .
It is importantly to note that cells are not simply a piece of elastic material. Their backbone, cytoskeleton, is a composition of tensile and compressive structures that integrate into an special architecture [16, 38]. The local change of mechanical forces will distort the cell shape as well as their cytoskeleton. On the other way around, the change of cell shape will also strengthen the accumulation of cellular stress by synthesizing or assembling additional bundles of tensile-stressed microfilament. Because cytoskeleton was remodeled when cells were forced to attach on cell-adherent islands, it explains why using cell-adherent islands with varied shapes is capable to provide mechanical stimulations to cells. In one of the early works, apoptosis, the cell "suicide" program, was trigger when cultured on smaller cell-adherent island, whereas proliferation was proportionally scaled with size of islands . However, it is not just because of the area of cell-adherent islands. The shape of cell spreading, which is associated with the internal stress, is likely a more critical factor than the contact area itself, given that DNA synthesis increases with the increase of cell projection area but not the summation of contact area (Fig. 1A, B). Similarly, when mesenchymal stem cells were cultured on islands with ether flower or star shape, the stress fiber, which is associated with the intensity of intracellular stress, accumulated at higher degree on star-shaped islands . This difference in turn resulted in different lineage commitment to either osteogenesis (bone tissue) or adipogenesis (fat tissue). Taking together, these results showed that cell shape, or the organization of the cytoskeleton, plays the leading role in cell mechanics, and can be easily controlled by microtechnology.
2.a.4 Preliminary results
As part of the PI's research background, we have identified that, in addition to the shape of individual cells, the differential adhesiveness for cell attachment is an important key for triggering the accumulation of mechanical stress [40, 41]. By culturing cells on alternating cell-adherent and cell-repellent stripes (Fig. 2A), the interface between cell-adherent and cell-repellent substrate triggers the accumulation of stress fiber, as shown by immunefluorescence of non-muscle myosin IIa (Fig. 2B-C). This cytoskeletal reorganization then induce an inherent left-right asymmetry that drives cells toÂ preferentially turn right on migration across the interfaces, eventually leading to multicellular structures as parallel aggregates align withÂ principal diagonal axis (Fig. 2D). By contrast, to test whether the stress fiber accumulation is induced by substrate interface or the inhomogeneous cell-cell contact at the edge of cell sheet, we used an additional method to restrict cell plating to specific regions. We used shadow mask plating, which creates stripes of cells and cell-free regions by plating cells through a stainless-steel mask that contains regularly spaced open windows allowing cells to be plated through. In this patterning technique, the stress fiber accumulation at the edge is absent (Fig. 2E-F), and cells failed to behave left-right biased migration, suggesting the presence of substrate interface is essential for the cellular stress accumulation and cytoskeletal remodeling. This finding was selected as the cover story in Circulation Research (Fig. 2G).
(b) Research plan and methodology
2.b.1 Overview of the research plan
Increased evidences showed that mechanical environment such as enriched extracellular matrix (collagen type I and fibronectin) is critical during tumor progression . This tissue remodeling produces denser and stiffer environment that enhances the cell proliferation and migration . More importantly, the changes of mechanical property could further lead to cell differentiation that enables the acquisition of ability to break away, unrestrictedly proliferate, invade into blood vessel, eventually become metastasis. To better understanding the mechanical factors in cancer physiology, this proposal aims at providing a novel microengineered platform which allows the development of new strategy for cancer therapeutics. Microtechnology such as photolithography will be used to fabricate chips with binary surfaces composed of cell-adherent and cell-repellent substrate (Fig. 3). Since the accumulation of mechanical stress depends on the level of cell spreading as well as the amount of substrate interface, different configuration of cell-adherent islands, e.g. circular, rectangular, and star shape, etc, will be implemented to provoke different level of mechanical stress in each cell. The induced stress will be quantified using stacked immunofluorescence image of stress fiber. For the larger cell-adherent island that is capable to lay down multiple cells, the intercellular communication will be recapitulated through cell-cell contact and its contribution can be determined. The biological functions such as viability, proliferation, metabolism, and migration in response to the mechanical condition will be assessed in our platform to obtain the physiological profile. Finally, based on the physiological profiles, we will select the population with highest potential for metastasis and seak the optimized therapeutic window.
Importantly, as compared to the macroscale forces that was applied to the entire culture, our microtechnology controls cell morphology with single-cell resolution, offering the capability to apply the specific, microscale force from one cell to another. Therefore, we can easily implement a huge variety of different shapes of cell-adherent islands, enabling high-throughput of case study and incorporate cell-cell contacts with specific modulation as desired. In addition, patterns without directional cue (cell-adherent islands as circles) can be studies to evaluate the 'quantity' of the applied stress without concerning the introduction of the 'gradient' of the applied stress as a byproduct. Also, this relatively simple design allows quantitative evaluation of the mechanical stress using immunofluorescence of stress fiber. As demonstrated in our preliminary results (Fig. 2C, F), stacking images provides statistic significance of the stress variation, and at the same time providing the enriched information as a two-dimensional mapping. Taking together, we envision the development of this platform will offer fast, informative, and inexpensive way which was not possible before.
2.b.2 Microfabrication of the cell-adherent and cell-repellent substrate
Photolithagraphy will be used to pattern a surface with either cell-adherent substrate, ECM, or cell-repellent substrate, polyethylene glycol (PEG). This protocol has been consistently used by the PI over the past 4 years. As shown in Figure 4, briefly, a glass substrate is cleaned and coated with hexamethyldisilazane (HMDS), followed by spin-coating with photoresist. The photoresist is exposed by ultraviolet, developed, and treated with oxygen plasma to remove the exposed HMDS. The remaining photoresist can be removed with acetone, IPA, and deionized water. For PEG coating, the HMDS/glass substrates are immersed in 3 mM of C3H9O3Si(C2H4O)6-9CH3 dissolved in anhydrous toluene with 1% triethylamine, followed by ultrasonication in anhydrous toluene, ethanol and deionized water . After drying, the HMDS/PEG substrates can be diced into chips and stored in vacuum desiccators. Prior to plating cells, the HMDS/PEG substrates are first incubated with diluted ECM solution in calcium-/magnesium-free phosphate-buffered saline. The protein-coated chip is then plated with cancer cells. After brief rinsing, only cells adhering to the ECM regions will be remained.
2.b.3 Cell types and culture
Given that over 90% of tumors are carcinomas, i.e. epithelial in origin, we will use non-metastatic mammary carcinoma cell line (67NR) and metastatic murine mammary carcinoma cells (EMT6) as our cancer model, with normal breast epithelial cell lines (MCF10A) as the control. 67NR cells will be cultured in high-glucose (4.5 mg/ml) Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% nonessential amino acids and 10% fetal bovine serum (FBS) . EMT6 cells will be cultured in alpha-minimal essential medium supplemented with 10% FBS . MCF10A cells will be cultured in 1:1 mixture of DMEM and Ham's F-12 media supplemented with 5% Horse Serum, 100 mg/mL streptomycin, 2 mM L-glutamine, and 20 mM HEPES, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone and 0.02 mg/ml EGF .
2.b.4 Quantification of cellular mechanical stress
The cytoskeletal remodelling reflecting the contractility is associated with the assembly of stress fiber  as well as the synthesis of primary motor protein, myosin IIa . To quantify the distribution of mechanical stress, we will use immunofluoscent staining where antibody will specifically label myosin IIa with fluoscent signal representing the amount of protein. Standard protocol will be used to carry out the immunofluorescence, as examplified in Fig. 5A. Briefly, cells cultured on the microengineered substrates are fixed in cold methanol (-20Â° C) to immobilize authentic subcellular structure and permit the access of antibody. The sample will then be blocked with blocking agent to prevent the non-specific binding. Next, cells will be incubated with solution of monoclonal Myosin-IIa antibody, followed by labeling of secondary antibodies to provide the fluorescent signal.
The fluorescence image will be acquired using inverted microscope with appropriate excitation wavelengths. To visualize the stress fiber distribution with better statistic significance, images will be stacked using etched microgrooves on the reverse surface for registration (Fig. 5B). After stacking images, the mechanical stress distributions were represented as the fluorescence intensity normalized by the number of images, and colored as the regular heatmap where red representing high value and blue representing low value (Fig. 2C, F).
2.b.5 Cell migration assay
Increased migration capability is the hallmark of metastasis. To test whether there is any association between migration rate and the increased mechanical stress, we will layout the cell-adherent patterns as horizontally stripes and measure the cell migration rate along the stripes (Fig. 6A). The applied mechanical stress will be controlled by the stripe width which determines the level of cell spreading. Importantly, since there will be no gradient of mechanical cue along the horizontal axis, the measured migration rate will truly reflect the influence of applied stress without being biased by other factors.
Time-lapse microscopy will be used to monitor the time-series of cell migration. The setup includes a microscopic thermal stage that maintains the culture condition at 37Â° C and continuously supplied with premixed 5% CO2. Previously we had demonstrated the adequacy of the on-stage incubator has been verified by the proliferation of NIH 3T3 fibroblast compared with that in a conventional incubator. Over 100 hours of culture, proliferation in the thermal stage remained comparable to that in the conventional incubator . For the measurement of cell migration, images will be acquired as a fixed interval using the inverted microscope in phase-contrast mode, and the migration rate will be determined by the migration trajectory versus the time, as an example shown in the Fig. 6B.
2.b.6 Detections of viability, proliferation and metabolism
The growth of cancer cell in response to the application of mechanical stimuli with/without chemical inhibitor will be evaluated by viability, proliferation, and metabolism. Before the assay, viability will be examined to avoid the over dosage of mechanical or chemical cues. To quickly discriminate live from dead cells, we will use the viabilityâ„cytotoxicity kit which simultaneously stains with green-fluorescent calcein-AM to indicate intracellular esterase activity (for live cells) and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity (for dead cells) (Fig. 7A).Â Within the adequate dosage window, cell proliferation will be evaluated by the synthesis of DNA using BrdUrd incorporation. BrdUrd is a syntheticÂ nucleosideÂ analogueÂ ofÂ thymidine. It can be incorporated into the newly synthesizedÂ DNAÂ of proliferating cells substituting for thymidine.Â Thus, after the exposure to BrdUrd-containing growth medium, cell will be fixed and stained using standard immunofluorescence protocol (similar to myosin IIa) targeting to BrdUrd (Fig. 7B), and the proliferation will be measured by the fluorescence intensity.
Cell metabolic activity will be measure by MTT assay. This colorimetric assay is based on the cellular enzyme activity that reduces the MTT, a tetrazolium dye, to an insoluble, purple formazan. After incubation, a solution of theÂ detergentsodium dodecyl sulfateÂ in dilutedÂ hydrochloric acid will be used to dissolve the insoluble purpleÂ formazan. Thus, the amount of formazanÂ product which indicates the level of metabolic activity can be measured by at 490 nm using a standard spectrophotometer.
2.b.7 Therapeutics treatment
Among the different conditions, the population with relatively high proliferation, metabolism, and migration capability will be selected given their highest potential for metastasis. For this selected cell population, the therapeutics treatment is to reduce the likehood of metastasis development, tumor size shrinking, and tumor growth. To reduce the proliferation which may be associated with application of mechanical stress, we will use the drugs that target to the proliferation and metabolism in rapidly growing cells. We will use fluorouracil (5-FU), which blocks the synthesis of pyrimidine thymidine, a nucleotide essential for DNA replication, finally causes cell cycle arrest and apoptosis (cell-suicide program). Also, we will apply folinic acid (leucovorin), which is frequently used in combination with 5-FU by enhancing 5-FU's inhibition of thymidylate synthesis. Additionally, the paclitaxel will also be applied. Paclitaxel arrests the microtubule breakdown by hyper-stabilizing the structure of microtubule such that it interferes with the normal microtubule breakdown during cell division.
Also, we will block the effects from cell mechanics by disorganizing cytoskeleton. Stress fiber accumulation is promoted by phosphorylation of myosin-II. Rho kinase up-regulates the phosphorylation of myosin-II and down-regulates myosin phosphatase (MYPT), which normally dephosphorylates myosin-II. Thus, inhibition of either myosin-II directly or Rho kinase leads to dissociation of stress fiber and the associated mechanical stress. Two types of small molecular, nonmuscle myosin II inhibitor (blebbistatin) [30, 40] and Rho kinase inhibitor (Y27632) [35, 40], will be used to intervene the signal pathway of actomyosin organization. In our past experience that studied the left-right asymmetry of cells (Fig. 2) , we found that in the presence of either inhibitor, results showed that stress fiber accumulation has been lost (Fig. 8A, B) and the multicellular aggregates failed to exhibit asymmetric alignment (Fig. 2D), instead, forming a labyrinthine pattern in the presence of Y27632 and a more disorganized pattern in the presence of blebbistatin (Fig. 8C, D). These results showed that stress fiber accumulation is required for the left-right biased cell migration, and demonstrated the efficacy of blebbistatin and Y27632 for the interference in cell mechanics.
2.b.8 Timeline and milestones
The PI has strong research experience utilizing microtechnology for studies in biological preparation , tissue morphogenesis , and cytoskeletal mechanics . Of note, the PI is one of the earliest researchers who identify the left-right biased cell migration depending on the mechanical stress provoked via substrate interface . This knowledge later leads to the engineering strategy for directing tissue morphology . Accordingly, the PI has the very strong confidence to effectively and efficiently accomplish the project in the proposed duration.
The proposed timeline and milestones is shown in Table 1. The project will begin from designing and fabricating the microchip for cell patterning. Meanwhile, the basic biological property of the cancer cell line such as normal proliferation rate, passage frequency, etc, will be cultivated and characterized. The stress fiber visualization and quantification will be carried out to elucidate the dependence between the stress distribution with the configuration of cell-adherent and cell-repellent substrate. The resulted knowledge will be fed back and iteratively optimize the configuration design. Also, the protocols for biological assays, such as proliferation, metabolism, and migration, will be developed to customize the needs for the specific cell line.
After the basic experience is gained, we will combine the therapeutic treatment with the mechanical stimuli to investigate the cell physiological profile and dosage window. Finally, with the different mechanical context, our platform will suggest the association between the mechanical conditions with the corresponding therapeutic dosage. Finally we will provide the strategy with the mechanical compartment for better cancer treatment.