Cells within tissues are continuously exposed to physical forces including hydrostatic pressure, shear stress, and compression and tension forces. Cells dynamically adapt to force by modifying their behaviour and remodelling their microenvironment. They also sense these forces through mechanoreceptors and respond by exerting reciprocal actomyosin- and cytoskeletal-dependent cell-generated force by a process termed 'mechanoreciprocity'. Loss of mechanoreciprocity has been shown to promote the progression of disease, including cancer. Moreover, the mechanical properties of a tissue contribute to disease progression, compromise treatment and might also alter cancer risk. Thus, the changing force that cells experience needs to be considered when trying to understand the complex nature of tumorigenesis.

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A Tense Situation: Forcing Tumour Progression

Cells sense their physical surroundings through mechanotransduction - that is, by translating mechanical forces and deformations into biochemical signals such as changes in intracellular calcium concentration or by activating diverse signalling pathways. In turn, these signals can adjust cellular and extracellular structure. This mechanosensitive feedback modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis, and is crucial for organ development and homeostasis. Consequently, defects in mechanotransduction - often caused by mutations or misregulation of proteins that disturb cellular or extracellular mechanics - are implicated in the development of various diseases, ranging from muscular dystrophies and cardiomyopathies to cancer progression and metastasis.

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Mechanotransduction gone awry.

Metastasis is a complex, multistep process responsible for >90% of cancer-related deaths. In addition to genetic and external environmental factors, the physical interactions of cancer cells with their microenvironment, as well as their modulation by mechanical forces, are key determinants of the metastatic process. We reconstruct the metastatic process and describe the importance of key physical and mechanical processes at each step of the cascade. The emerging insight into these physical interactions may help to solve some long-standing questions in disease progression and may lead to new approaches to developing cancer diagnostics and therapies.

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The physics of cancer: the role of physical interactions and mechanical forces in metastasis

Mechanical interactions are fundamental to biology. Mechanical forces of chemical origin determine motility and adhesion on the cellular scale, and govern transport and affinity on the molecular scale. Biological sensing in the mechanical domain provides unique opportunities to measure forces, displacements and mass changes from cellular and subcellular processes. Nanomechanical systems are particularly well matched in size with molecular interactions, and provide a basis for biological probes with single-molecule sensitivity. Here we review micro- and nanoscale biosensors, with a particular focus on fast mechanical biosensing in fluid by mass- and force-based methods, and the challenges presented by non-specific interactions. We explain the general issues that will be critical to the success of any type of next-generation mechanical biosensor, such as the need to improve intrinsic device performance, fabrication reproducibility and system integration. We also discuss the need for a greater understanding of analyte–sensor interactions on the nanoscale and of stochastic processes in the sensing environment.

Comparative advantages of mechanical biosensors

Cancer initiation and progression follow complex molecular and structural changes in the extracellular matrix and cellular architecture of living tissue. However, it remains poorly understood how the transformation from health to malignancy alters the mechanical properties of cells within the tumour microenvironment. Here, we show using an indentation-type atomic force microscope (IT-AFM) that unadulterated human breast biopsies display distinct stiffness profiles. Correlative stiffness maps obtained on normal and benign tissues show uniform stiffness profiles that are characterized by a single distinct peak. In contrast, malignant tissues have a broad distribution resulting from tissue heterogeneity, with a prominent low-stiffness peak representative of cancer cells. Similar findings are seen in specific stages of breast cancer in MMTV-PyMT transgenic mice. Further evidence obtained from the lungs of mice with late-stage tumours shows that migration and metastatic spreading is correlated to the low stiffness of hypoxia-associated cancer cells. Overall, nanomechanical profiling by IT-AFM provides quantitative indicators in the clinical diagnostics of breast cancer with translational significance.

https://www.cell.com/biophysj/pdf/S0006-3495(12)03025-1.pdf

The nanomechanical signature of breast cancer

Change in cell stiffness is a new characteristic of cancer cells that affects the way they spread1,2. Despite several studies on architectural changes in cultured cell lines1,3, no ex vivo mechanical analyses of cancer cells obtained from patients have been reported. Using atomic force microscopy, we report the stiffness of live metastatic cancer cells taken from the body (pleural) fluids of patients with suspected lung, breast and pancreas cancer. Within the same sample, we find that the cell stiffness of metastatic cancer cells is more than 70% softer, with a standard deviation over five times narrower, than the benign cells that line the body cavity. Different cancer types were found to display a common stiffness. Our work shows that mechanical analysis can distinguish cancerous cells from normal ones even when they show similar shapes. These results show that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.

Nanomechanical analysis of cells from cancer patients

Recently biomechanics of cancer cells, in particular stiffness or elasticity, has been identified as an important factor relating to cancer cell function, adherence, motility, transformation and invasion. We report on the nanomechanical responses of metastatic cancer cells and benign mesothelial cells taken from human body cavity fluids using atomic force microscopy. Following our initial study (Cross et al 2007 Nat. Nanotechnol. 2 780-3), we report on the biophysical properties of patient-derived effusion cells and address the influence of cell morphology on measured cell stiffness. Using a cytocentrifugation method, which yields morphologically indistinguishable cells that can be prepared in 1 min and avoids any possible artifacts due to 12 h ex vivo culture, we find that metastatic tumor cells are more than 80% softer than benign cells with a distribution over six times narrower than that of normal cells. Consistent with our previous study, which yielded distinguishable cell populations based on ex vivo growth and morphological characteristics, our results show it is unlikely that morphology alone is sufficient to explain the difference in elastic moduli for these two cell types. Moreover, analysis of non-specific cell adhesion inherent to tumor and normal cells collected from patients show surface adhesion of tumor cells is ∼33% less adhesive compared to that of normal cells. Our findings indicate that biomechanical-based functional analysis may provide an additional platform for cytological evaluation and diagnosis of cancer in the future.

https://iopscience.iop.org/article/10.1088/0957-4484/19/38/384003/pdf

AFM-based analysis of human metastatic cancer cells

Applicability of AFM in cancer detection

This study used atomic force microscopy (AFM) to probe the local cell-surface interactions associated with the glucan polymers of Streptococcus mutans, the macromolecules most commonly attributed to the virulence of this microbe. In situ force spectroscopy was used to quantitatively probe and correlate cell-surface adhesion and dynamics with S. mutans UA140 wild-type and five glucosyltransferase mutants. Adhesion between the tooth surface and S. mutans is largely mediated by glucan production from sucrose via three glucosyltransferases (Gtfs; GtfB, GtfC and GtfD). To monitor the contribution of these particular Gtfs, isogenic mutants of S. mutans were constructed by specific gene inactivation and compared to the wild-type under sucrose and non-sucrose conditions. We report direct measurement of the mechanical properties associated with glucan macromolecules demonstrating that the local adhesion strength increases in a time-dependent process, with a decrease in the average number of rupture events. This finding suggests that S. mutans attaches mainly through glucans to surfaces in the presence of sucrose. In addition, a possible role of the Gtf proteins in sucrose-independent attachment is supported by the decreased adhesion properties of the GtfBCD mutant compared to the wild-type.

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Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions

Green tea extract (GTE) is known to be a potential anticancer agent (Yang et al 2009 Nat. Rev. Cancer 9 429-39) with various biological activities (Lu et al 2005 Clin. Cancer Res. 11 1675-83; Yang et al 1998 Carcinogenesis 19 611-6) yet the precise mechanism of action is still unclear. The biomechanical response of GTE treated cells taken directly from patient's body samples was measured using atomic force microscopy (AFM) (Binnig et al 1986 Phys. Rev. Lett. 56 930). We found significant increase in stiffness of GTE treated metastatic tumor cells, with a resulting value similar to untreated normal mesothelial cells, whereas mesothelial cell stiffness after GTE treatment is unchanged. Immunofluorescence analysis showed an increase in cytoskeletal-F-actin in GTE treated tumor cells, suggesting GTE treated tumor cells display mechanical, structural and morphological features similar to normal cells, which appears to be mediated by annexin-I expression, as determined by siRNA analysis of an in vitro cell line model. Our data indicates that GTE selectively targets human metastatic cancer cells but not normal mesothelial cells, a finding that is significantly advantageous compared to conventional chemotherapy agents.

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Sarah E. Cross

Papers

Nanomechanical analysis of cells from cancer patients

AFM-based analysis of human metastatic cancer cells

Applicability of AFM in cancer detection

Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions

Green tea extract selectively targets nanomechanics of live metastatic cancer cells