AFM nanoindentation detection of the elastic modulus of tongue squamous carcinoma cells with different metastatic potentials

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Probing mechanical properties to study cancer cell migration

To best comprehend cellular behaviour and how it determines cell migration in metastatic cancer, the research described here has focused on cell mechanics. The signalling pathway involving Rho-associated kinase (ROCK) has emerged as being the main regulator for the cellular cytoskeleton and actomyosin contractility that play key roles in metastatic cancer formation. In this thesis, an examination is made of how the cellular properties intertwine as ROCK is overexpressed. In research towards being able to measure and describe the viscoelastic properties of a cell that are associated with cell mechanics, over a wide range of timescales, a novel AFM force indentation data analysis method was applied. In particular, as part of this study, pancreatic ductal adenocarcinoma (PDAC) cells were overexpressed with ROCK, and the influence of ROCK activity on cell’s elastic and viscoelastic properties were quantified. It was found that when ROCK activity was overexpressed in cells, their elasticity decreased while their viscosity remained unchanged. These properties had a direct correlation with the activity of ADF/cofilin - the proteins downstream of ROCK. This meant that with overexpression, more stable actin bundles were present along with their inward stresses generated by the actomyosin contraction. This is consistent with an increased level of compressive forces within cells. Collective compressive forces between cell-cell are associated with the packing of cells that decreases cellular response. To further understand the role of ROCK activity in cancer invasion, a microfluidic device was created to mimic cell migration through tissue. The device consists of precisely defined microchannels with dimensions chosen to hinder and confine the cells in a manner similar to that found in a physiological environment. It was found that overexpressed ROCK1 cells in the confinement had notable decrease in cell size and motility. Along with this decrease in mechanical properties, observations also gave rise to questions about the connection between these properties that remain to be answered

Investigation of stiffness as a biomarker in ovarian cancer cells

In this dissertation, we developed cell stiffness as a biomarker in ovarian cancer for the purpose of grading metastatic potential. By measuring single cell stiffness with atomic force microscopy and quantifying in vitro invasiveness of healthy and cancerous ovarian cells, we demonstrated that cancerous ovarian cells have reduced stiffness compared to the healthy ones and invasive ovarian cancer cells are more deformable than noninvasive ovarian cancer cells. The difference in cell stiffness between two genetically similar cell lines was attributed to actin-mediated cytoskeletal remodeling as revealed by comparative gene expression profile analysis, and was further confirmed by fluorescent visualization of actin cytoskeletal structures. The actin cytoskeletons were innovatively quantified and correlates with cell stiffness distributions, further implicating actin-mediated cytoskeletal remodeling in stiffness alteration from the perspective of structure-property relationship. The correlation between stiffness and metastatic potential was also demonstrated in pancreatic cancer cell line AsPC-1, which shows reduced invasivess and increased stiffness upon treatment with N-acetyl-L-cysteine (NAC), a well known antioxidant, reactive oxygen species (ROS), scavenger and glutathione precursor. The correlation between cell stiffness and metastatic potential as demonstrated in ovarian and pancreatic cancer cells indicated that mechanical stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells, and might be useful clinically with the development of rapid biomechanical assaying techniques. We have also investigated the stiffness evolution through progression of the cell cycle for the healthy ovarian phenotype and the invasive cancer ovarian phenotype, and found that the healthy phenotype at G1 phase are significantly stiffer than other single cells except the invasive phenotype at late mitosis; other groups are not significantly different from each other. We have also investigated intracellular heterogeneity and mechanical nonlinearity in single cells. To this end, we developed a methodology to analyze the deformation-dependent mechanical nonlinearity using a pointwise Hertzian method, and tested the method on ultrathin polydimethylsiloxane (PDMS) films which underwent extremely large strains (greater than 50%). Mechanical stiffening due to large strain and geometrical confinement were observed. The onset of nonlinearity or mechanical stiffening occurs at 45% of the film thickness, the geometry induced stiffening causes an increase in stiffness which shows a strong power law dependence on film thickness. By applying the pointwise Hertzian method on stiffness measurements with AFM that were collected on living cells, we also investigated the nonlinear and heterogeneous mechanics of single cells, since attachment of cells to stiff substrate during indentation may impact their mechanical responses. Even under natural biological conditions, cells confined in narrow spaces may experience heightened mechanical stiffness. Through indentation-dependent force mapping, analysis of the local cell stiffness demonstrated spatial variation. The results indicated that the mechanical properties of single cells are highly nonlinear and are dependent upon the subcellular features under the applied force as well as the dimensions of the cellular material. We identified single cell stiffness as a potential biomarker of the metastatic potential in ovarian cancer, and quantified the effect of geometrical confinement on cell mechanics. The results presented in this dissertation not only made contributions to the development of accurate, non-invasive clinical methods to estimate metastatic potential of ovarian and perhaps other types of cancer, but also shed light on the intracellular mechanical information by developing new techniques to quantify the effect of geometry on cell mechanics.Ph.D

Relating Mechanical and Genetic Data at Single Cell Level across the Genome to Investigate Metastasis

Nine out of every ten cancer-related deaths is caused by metastasis, but the molecular mechanisms driving this process are still not fully understood. Several studies have implicated that as a cell’s metastatic potential increases, cell stiffness decreases. Yet while certain genes that affect cell mechanics have been studied, a genome-wide study of networks that modulate cell biophysical properties has not been attempted. The long-term goal of this research is to understand the molecular and mechanical mechanisms driving metastasis. To reach this goal, a new methodology was developed to combine mechanical and gene expression data for the same single cells. Additionally, a novel microfluidics approach for cell sorting based upon biophysical properties was leveraged for the high-throughput discovery of genes linked to cell mechanics and metastasis. These approaches led to deeper understanding of how cellular mechanics are regulated within the context of networks of genes associated with increased metastatic potential. I investigated this intersection through the following aims: 1) Create and validate a combined single cell mechanics and gene expression methodology, 2) Identify genes related to mechanical changes in cancer cells through GeCKO high-throughput mechanical screen, and 3) Validate phenotypic and mechanotypic importance of genes of interest.Ph.D

Role of Syk in the regulation of cytoskeleton and stress granules in breast cancer

The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. Treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk tothe stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus

Tuneable 3D biocompatible scaffolds for biological and biophysical solid-tumour microenvironment studies; applications in Ovarian Cancer

Recently, three-dimensional (3D) tumour models mimicking the tumour microenvironment and reducing the use of experimental animals have been developed generating great interest to appraise tumour response to treatment strategies in cancer therapy. As tumours have distinct mechanics compared to normal tissues, biomaterials have also been utilized in 3D culture to model the mechanical properties of the tumour microenvironment, and to study the effects of extracellular matrix (ECM) mechanics on tumour development and progression. Mechanical cues regulate various cell behaviours through mechanotransduction, including proliferation, migration, and differentiation. In the context of cancer, both stromal cells (cancer associated fibroblasts) and tumour cells remodel the ECM and change its mechanical properties, and the altered mechanical niche in turn is likely to influence tumour progression. In this study, bovine derived collagen type I and Jellyfish derived marine collagen sources, were tested as biomaterial candidates for cancer studies, moulded to porous scaffolds with tuneable mechanical properties. The resulting interconnected network of collagen fibre constructs, fabricated using lyophilisation provide good control of scaffolding architecture, pore sizes range, high porosity levels, high level of cell viability and low production cost. Importantly these sponge scaffolds were, in the form of 3D models, compatible with a host of cellular and molecular biology assays used to investigate mechanical and biological effects of collagen crosslinking and (hyaluronic acid) HA inclusion on both fibroblasts and ovarian cancer cells. Stromal cells and cancer cells respond differently to the altered stiffness of their local microenvironment. Fibroblasts, once activated with TGF1, converge toward a ‘senescent-like phenotype’, blocking migration and matrix remodelling and promote tumour progression, probably through the secretion of tumour-promoting signals, in stiffer mechanical environments. Cancer cells, of both epithelial and mesenchymal phenotype, respond to increased local matrix stiffness by increasing proliferation while, at the same time, becoming more susceptible to treatment. Mechanically informative scaffolds resemble the physical characteristics of both normal and pathological ovarian tissue mechanics, where ovarian cancer originates. Physical changes observed in the later stage of ovarian cancer disease progression may therefore be fundamental for the increased cancer proliferation that drives metastatic progression, however opening an interesting window for cancer treatment. Bio-physical inclusive models not only lead the path to unveil complex interactions of biophysical and biological signals in the tumour microenvironment, but they represent a highly informative and effective platform to test novel target therapies with effective costs and high throughput. They can accommodate coculture systems and potentially patients-derived cell cultures, providing a platform to test current and new drugs and to evaluate drug efficacy following a precision medicine approach

Cell Mechanics in Physiology: A Force Based Approach

All biological systems rely on complex interactions with their external and internal environments where the key factors are force sensing and force generation. These systems are highly dynamic, and recent studies have shown that it is the control and maintenance of these interactions that are essential for normal functioning. Appreciation of these roles has led to a revolution in instrumentation and techniques to study and model mechanical interaction at all length and time scales in biology. The work presented here is one such effort, utilizing a magnetics based force system to study and understand the mechanisms of cell mechanics and their role in mucuciliary clearance in the lung and in cancer cell invasion and metastasis. I first introduce the instrumentation and describe basic rheological concepts that govern the study of cell mechanics. I then report on the application of this system to study the force generation and dynamics of airway cilia. The bulk of the work is focussed on the role of cytoskeleton mechanics in cancer. I present our results which show the remarkable relationship between the cell's mechanical properties and its metastatic potential. Finally, I report on a novel pathway which is responsible for force mediated sensing in cells and show that this pathway is deregulated in cancer. These results have strong implications on the potential of stiffness and force sensing pathways as novel cancer therapeutic targets

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome

Quartz Crystal Microbalance with Dissipation Monitoring: Sensing Beyond Cell-Substrate Adhesion

The quartz crystal microbalance with dissipation monitoring (QCM-D) is an ultrasensitive mechanical sensing device that is capable of providing real-time, non-invasive measurements of changes in resonance frequency and energy dissipation responses of cells immobilized onto the sensor surface. The majority of its applications in cell research have been limited to the study of the adhesive interaction between cells and the substrate surface and the evaluation of the effect of an external stimulant on the adhered cells. The overall objective of this thesis work was to further exploit the capabilities of the QCM-D in cell research by addressing important problems that are relevant to fundamental biology and medicine. In the project presented in Chapter 4, we examined the EGF-induced cell de-adhesion, a critical step in normal embryonic development, wound repair, inflammatory response, and tumor cell metastasis. We were able to successfully establish the change in the energy dissipation factor ([delta]D-response) as a specific and quantitative measure of cell adhesion. With this novel measure of cell adhesion, we characterized this complex de-adhesion process, which appeared to exhibit an initial rapid cell de-adhesion, a transition, and a slow re-adhesion. We also shed light on the dynamic coordination of the three downstream pathways of epidermal growth factor receptor (EGFR) signaling in mediation of the epidermal growth factor (EGF)-induced de-adhesion process. In chapter 5, continuing with the theme of applying this novel measure to the characterization of cell adhesion, we examined the adhesion process of human epidermal keratinocytes on the implant type of surface. We identified three distinct stages of this adhesion process and developed several new strategies for strengthening the adhesion between soft tissue/skin/bone and implants. In chapter 6, we extended this novel measure of cell adhesion to the investigation of GPCR signaling by capitalizing the regulatory role of G protein-coupled receptor (GPCR) signaling in mediation of cell adhesion. We were able to dissect the multiplicity of the ligand-induced GPCR signaling and obtain mechanistic insights into the promiscuous coupling of G[alpha]q, G[alpha]s, and G[alpha]i pathways as well as their dynamic coordination. In chapters 7 and 8, we explored the potential of cell-based QCM-D assay in detection of biomarkers. In chapter 7, we were able to relate the [delta]D-response with the cellular response mediated by the high-affinity EGFR, the subclass of EGFR that is more relevant to cancer development. Lastly in chapter 8, we demonstrated that this cell-based QCM-D assay has the sensitivity and specificity to detect some of the potential biomarkers of ovarian cancer. In conclusion, this thesis work has demonstrated that the QCM-D is a highly sensitive, label-free technique that has the capabilities to probe some of the most important cellular processes, such as cell adhesion and cell signaling and to serve as a sensing platform for biomarker detection.Ph.D., Chemistry -- Drexel University, 201

Biophysical and biomolecular analysis of EVs and their interaction with target cells

Triple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtype and with a poor prognosis. Nowadays, chemotherapy is the main treatment in both early and advanced stage of the TNBC, but patients without complete response to conventional chemotherapy are approximately 80%. In light of that, clarifying biological mechanisms of the metastatic process is crucial in finding new therapeutic approaches for effective interventions. Metastasis is thought to be easier for more deformable and, therefore, soft cancer cells, which can migrate through narrow pores of extracellular matrix and vessels. Extracellular vesicles derived from triple-negative breast cancer, by sharing oncogenic molecules, have been shown to promote proliferation, drug resistance migration and metastatic capability in target cells proportional to properties of donor ones. Considering all these evidence, we wondered if small-EVs could also transfer information to target cells about biomechanical properties, a key step in metastasis, of the cell from which they originate. Our results showed that small-EVs derived from the MDA-MB-231 cell line (TNBC) can directly modulate biomechanical properties (stiffness/Young\u2019s modulus), cytoskeleton, nuclear morphology and Yap activity of MCF7 cell line (Luminal A) as target cell. Therefore, in this study, we found out a new mechanism through which small-EVs derived from TNBC subtype could be able to contribute to progression and metastatic processes in breast cancer; this new knowledge could be used in diagnostic and therapeutic field