Brief Description of Cancer Cells

Figure 1. Cell growth for normal and cancer cells. (a) A schematic showing normal cell division. In this case a damaged cell beyond repair undergoes apoptosis, a process of programmed cell death (or “cell suicide”). This process plays a role in tumor suppression. (b) Schematic of cancer cell division showing loss of normal growth control. In cancer cell division, mutations occur and are proliferated resulting in uncontrolled growth of mutated cells. Damaged (mutated) cells no longer experience programmed cell death (apoptosis); inhibition of apoptosis is a hallmark of cancer.

Figure 2. Cancer cell invasion and metastasis. (i) Cancer cell invasion of surrounding tissue. (ii) Angiogenesis (the formation of new blood vessels from pre-existing blood vessels) and cancer cell transportation via the circulatory system. (iii) Cancer cell metastasis (spreading from a primary site) and growth at a new location. Image courtesy of the National Cancer Institute (NCI).

Brief Description of Atomic Force Microscopy (AFM)

A cell is a basic unit of life with very accurate control of functions such as synthesis, sorting, storage and transport of biomolecules; the expression of genetic information; signal transduction; the response to changes in external environment; and the powering of molecular motors and machines[1]. The mechanics involved in many of these processes are pivotal for understanding the structure-function relationships, organization and regulatory mechanisms associated with cells[2]. Recently, considerable progress has been made in probing the mechanics associated with single cells and biomolecules coupled to the cell membrane[3,4]. In particular, atomic force microscopy (AFM) has emerged as an important instrument for the investigation of mechanical properties associated with live cells[3,4,6,7,8,9].

AFM has come to be regarded as a powerful tool for probing biological samples with sub-nanometer resolution thus providing tremendous insight regarding the surface features and cellular nanomechanics, or cellular processes based on the mechanical properties of living cells[10]. An AFM consists of a cantilever (with tip mounted to the soft cantilever spring), a sample stage and an optical beam deflection system which consists of a laser diode and a position sensitive photodiode (Fig. 3). A schematic diagram of an AFM tip interacting with an individual cell is shown in Figure 4. Mechanical measurements acquired using AFM rely on measuring the force as the tip is pushed toward (Figure 4a), indented into (Figure 4b) and retracted from the sample or cell surface in this case (Figure 4c). The cantilever is mounted on the end of a piezoelectric tube scanner (PZT) which is used to bring the tip into contact with the surface. The force is measured by recording the deflection (vertical bending) of the cantilever. As described above, the cantilever deflection is usually detected by a laser beam focused on the free end of the cantilever and reflected into a photodiode; this deflection is directly proportional to the force. Force-displacement curve are obtained by monitoring the deflection of the cantilever (as described in Fig. 4). The micro-cantilever based system allows us to probe the local Young’s modulus (E) or “stiffness” of living cells, perform force spectroscopy measurements with pico Newton resolution, and provides a sensor to record in vivo measurements of the cell wall at sub-nanometer resolution. In particular, AFM is a key tool in acquiring kinetic information, and real-time signals of living cells, and is capable of offering in vivo single-cell diagnostics. AFM measurements generate a greater understanding of structure, function, and relationships of biological macromolecules, thus generating characteristics inherent to specific biological cells[13]. These emerging concepts aid in the development of new types of nanomechanical sensors, which may contribute significantly to the understanding of changes in cytoarchitecture, which are characteristic of cellular dedifferentiation, malignant transformation, growth activation, cell motility and disease states, such as that noted in our manuscript titled “Nanomechanical analysis of cells from cancer patients’” published in Nature Nanotechnology (Cross et al. 2007).  The use of AFM to probe and study single biological systems on the nanoscale can yield information about the integrity and local nanomechanical properties of these cells[14]. The AFM approach may help us to elucidate the mechanics inherent to changes in cytoarchitecture and dynamics under in vitro conditions and may help elucidate the mechanisms and related biological alterations associated with tumor phenotype. 

Figure 3. A schematic of a basic AFM set-up. Using an optical beam deflection system, the position of the AFM cantilever tip is recorded as the tip is brought into contact with the surface (in this case, the cell’s surface). A cantilever is mounted onto the end of a piezoelectric tube scanner (PZT) which is used to bring the tip into contact with the surface. A laser diode is focused on the back of an AFM cantilever and the beam is reflected into a position sensitive photodiode. Mechanical properties and interaction forces of a surface (or cell surface) can be determined from force-displacement curves recorded from the deflection of the cantilever (see Fig. 4 for more detail).

Figure 4. Schematic of an AFM tip inter-acting with a cell surface. (a) AFM tip approaching cell surface, (b) indented into cell surface and (C) retracted from cell surface. The central inset shows a schematic of a force-displacement curve (a-c corres-pond to the positions described above), which is recorded as the “approach” and “retract”curves of the cantilever as it moves toward and away from the surface. The force acting on the cantilever is recorded as a function of the piezoelectric crystal displacement (as described in Fig. 1). Mechanical properties, such as the Young’s Modulus, E, or cell stiffness, can be calculated from force curves using a Hertz model[11,12].

Significance

Cytology in general is a low cost, non-invasive or minimally invasive technical for cancer diagnosis clinically.   However, conventional morphological analysis using light microscopy is limited by poor sensitivity and accuracy.  New technologies will be needed to overcome such a problem.  AFM-based cytological analysis provides an entirely new technique platform for cancer diagnosis and evaluation, and we hope our study will help facilitate the translation of this technology into a clinical setting. 
Whereas current tests may be able to distinguish metastatic adenocarcinoma from mesothelial cells, such tests may not be utilized to distinguish other types of metastatic cancers such as squamous cell carcinoma, melanoma, and sarcoma. Furthermore, there is no test available so far to accurately diagnose malignant mesothelioma on a body cavity fluid cytology specimen, which by cytomorphology alone may be extremely challenging. This again argues for additional tests, such as nanomechanical analysis using AFM, which can be used to aid the diagnosis in body cavity fluid samples.

Glossary of Select Terms

Benign – characteristic of a tumor that does not invade surrounding tissue or will not metastasize.
Carcinoma – a malignant tumor of the epithelium.
Effusion – a collection of effused fluid.
Malignant – characteristic of a tumor that is capable of invading surrounding tissue and of metastasizing to secondary locations.
Metastasis – the process of cancer cells spreading from a primary tumor site to secondary sites in the body.
Morphology – the study of the form, shape and structure of a cell.
Phenotype – the observable characteristics of a cell.
Pleural effusion – the fluid accumulated between the layers of membranes that line the lungs and chest cavity.
Prognosis – a forecast or future outlook of a disease.

References

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