Cellular Analysis by Atomic Force Microscopy

Malgorzata Lekka

• Publisher: Jenny Stanford Publishing
• Year: 2017
• ISBN:
  9781315341156
• Edition: 1
• Topic: Medicine
• Subtopic: Oncology

Despite substantial evidence showing the feasibility of Atomic Force Microscopy (AFM) to identify cells with altered elastic and adhesive properties, the use of this technique as a complementary diagnostic method remains controversial. This book is designed to be a practical textbook that teaches how to assess the mechanical characteristics of living, individual cells by AFM. Following a step-by-step approach, it introduces the methodology of measurements in the case of both determination of elastic properties and quantification of adhesive properties.

Chapter 1



Introduction

Cancer is a very complex disease, involving multiple molecular and cellular processes arising from a gradual accumulation of genetic changes in individual cells. The most apparent morphological change is visible during the transition from a benign tumor to metastatic tumors where cells alter from highly differentiated normal phenotypes to migratory and invasive ones. Around 90% of all cancer deaths are due to metastatic spread of primary tumors. The criteria utilized to detect cancerous cells have been mainly relying on biological and morphological description, additionally complemented by a variety of other techniques, including genetic, chemical, and immunological methods, applied in order to fine-tune diagnosis or therapy. Despite enormous efforts to develop better treatment protocols, our ability to cure solid tumors, such as those of the breast, prostate, cervix or colon, is still lacking sufficient detection methods [1].

The cells transformed oncogenically differ from normal ones in many ways, encompassing variations in any cellular aspects such as growth, differentiation, interactions between neighboring cells and/or with the extracellular matrix (ECM), cytoskeleton organization, and several others. Poor differentiation of the cytoskeleton can result in the larger deformability of cancerous cells. Low stiffness of cancer cells is related to a partial loss of actin filaments and/or microtubules, and therefore by lower density of the cellular scaffold [2, 3]. Moreover, one of the key phenomena in metastasis includes adhesive interactions, maintained by distinct type of adhesion molecules present on a cell surface. Cancerous cell aptitude for invasion and migration (clinically interpreted as tumor aggressiveness) has been associated with poor differentiation of the cell and the altered adhesive interactions that characterize a vast majority of cancer cells.

Cellular Analysis by Atomic Force Microscopy

Malgorzata Lekka

Copyright © 2017 Pan Stanford Publishing Pte. Ltd.

ISBN 978-981-4669-67-2 (Hardcover), 978-1-315-36480-3 (eBook)

www.panstanford.com

It is obvious that novel techniques are in the limelight if they are able to bring more precise, local information about cancerous changes as early as possible. There are rather few methods capable to assess cell mechanical properties. Historically, the first technique was the micropipette aspiration [4, 5]. Other researchers have employed the magnetic bead rheology [6], microneedle probes [7], acoustic microscopes [8], and the manipulation of beads attached to cells with optical tweezers [9, 10]. Among these techniques, the atomic force microscopy (AFM) can detect malignant changes with a very high resolution, being applied either in imaging mode or as the technique providing information about the mechanical properties of living cells (i.e., their ability to deform and to adhere) in a quantitative manner. Its main advantage is the possibility to measure biological objects directly in their natural environment, such as buffer solutions or culture media.

Many publications in this area were devoted to the characterization of single cells’ deformability and adhesiveness, presented in a broad context of biological targets, starting from cell motility, would healing, muscle contraction or differentiation and ending up in characterization of various pathologies such as muscular dystrophies, blood diseases or cancers. Therefore, in this Chapter, the importance of cellular ability to deform and to adhere is presented with the focus on the AFM-related aspects in cancer studies.

1.1 Cell Ability to Deform

Within the past two decades, the cellular ability to deform has attracted great interest in the field of biology. This is because in human body, various cell types are continuously exposed to passive (stretch, compression) and/or active (contraction) deformations. The technological development of techniques, that enable to probe elastic properties of single cells, has been shown to be more powerful than that of bulk measurements since the former ones can relate the mechanical properties with cellular functioning and structures.

The capability of cells to deform has been studied long time ago. One of the earliest reports of increased deformability of cancerous cells has been reported by Ochalek et al. [11]. In these studies, the microfiltration experiments were used to study the migration capability of B16 mouse melanoma cells. In the filtration experiment, the assumption was that all melanoma cells capable to metastase passed through the filter. This was justified by the condition that metastatic cells must be squeezed to go through the surrounding tissue matrix when they make their way into the circulatory systems where they are directed to establish distant settlements. However, the conclusion has been built by counting cells and quantifying the filtration time, not by the determination of cells mechanical properties. The pioneering study [12] showed the importance of mechanical properties to characterize cancerous cells. In these studies, the deformability of human bladder cancerous cells (cell lines: T24, Hu456, BC3726) was one order of magnitude larger than for their reference counterpartners (cell lines: Hu609, HCV29). These early results have been supported (and indirectly verified) by optical tweezers measurements. Using this latter, high throughput technique, three cell lines were compared, namely, a non-tumorigenic breast epithelial MCF10 cells, a non-motile, non-metastatic breast epithelial cancer MCF7 cells, and MCF7 cells transformed with phorbol ester causing the increase in the cancer cell invasiveness. The results showed significant increase of MCF7 cells deformability compared to MCF10 and non-transformed MCF7 ones [10].

Based on single-cell deformability measurements, it has been found that cell structure is closely related to specific mechanical properties, which, in turn, depend on the organization of cell cytoskeleton. The role of cytoskeletal components (mainly actin filaments and microtubules) in cellular deformability has been shown by applying so-called cytoskeletal drugs that influence the structure and formation of each particular component [13, 14, and 15]. For example, cytochalasin D increases the cellular deformability while nocodazol leads to cell stiffening. A summary of cytoskeletal drugs influence on cellular deformability is presented in Table 1.1. Depending on the type of the compounds, disrupting or stabilizing particular cytoskeletal elements, the influence on cellular deformability manifests either in higher or in lower deformability (cells become softer or more rigid, respectively). However, it should be underlined that the effect is dependent on the applied concentration and time of action.

Table 1.1 The effect of common cytoskeletal drugs on cellular deformability

Drug	Known effect	Effect on cellular mechanics
Cytochalasin B	Disrupt actin filaments (F-actin) Disassembled stress fibers	Increased deformability
Cytochalasin D	Disrupt actin filaments (F-actin) Disassembled stress fibers Aggregation of actin within the cytosol	Increased deformability
Latrunculin A	Disrupt actin filaments (F-actin) Disassembled stress fibers	Increased deformability
Jasplakinoide	Disrupt actin filaments (F-actin) but did not disassemble stress fibers	Increased deformability (cell becomes softer)
Colchicine	Disrupt microtubules	No effect
Colcemid	Disrupt microtubules	No effect or increased deformability
Taxol (paclitaxel)	Stabilize microtubules	No effect or decreased deformability
Nocodazol	Stabilize microtubules	No effect or decreased deformability

The cytoskeleton interaction with associated proteins has been demonstrated to influence cellular elastic properties for cells expressing vinculin (a focal adhesion protein interacting with actin fibers). The loss of vinculin reflects in a noticeable reduction of cell adhesion, spreading and the presence of stress fibers. The comparison performed by Goldman et al. in 1998 showed that the vinculin-deficient F9 mouse embryonic carcinoma cells had lower Young’s modulus than the wild-type cells. The authors attributed these changes to altered actin cytoskeletal organization, indicating an important role of vinculin as an integral part of the cytoskeletal network [16].

In the AFM technique, the deformability is expressed by Young’s modulus value which delivers a quantitative measure of cellular elasticity. It is a very local feature, showing usually large discrepancy when measured on a single cell as well as within a population of cells. The latter variations are attributed to heterogeneity of cellular structures, while the former reveal a non-uniformity of cell populations. It has been reported that cells in vitro have Young’s modulus values in the range of 1–100 kPa [17, 18], which encompasses different types of investigated cells, including vascular smooth mu...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Contents
• Preface
• 1. Introduction
• 2. Cell Structure and Functions
• 3. Principles of Atomic Force Microscopy
• 4. Quantification of Cellular Elasticity
• 5. Adhesive Properties Studied by AFM
• 6. Conclusions
• Index