The History About Extracellular Matrix

Published Date: 02 Nov 2017

The aim of this project is to assess if manipulating the extracellular environment alters the types of structures we normally get with the human breast cell line MCF10A, using synthetic hydrogel route. If by adding inert hydrogel at a level which mimics the stiffness of Matrigel we get a similar proportion of acini to ducts then the effect of morphogenesis may be largely biophysical.

2. Introduction

The human breast tissue is made up of connective tissue and gland tissue divided into lobes. Breasts individually contain between fifteen to twenty individual lobes which branch from the nipple (Krause 2009). The lobes are separated via dense connective tissue. Individual lobes are separate mammary glands and contain glandular tissue which are drained by their own channel of components fixed within connective tissue (Krause 2009).Breast tissue is made up of acini, collagen and ducts. Acini are a cluster of cells found within a gland (Medical Dictionary 2013) Collagen is made up of protein fibres known as fibril. It gives the breast its stiffness. (Medical Dictionary 2013). Figure 1 below shows the anatomy of the breast.

Figure 1- Anatomy of the Breast (Clinical Symposia Volume 32 number 2, 1980, breast lumps, CIBA)

1.2 Cell Lines

Cell lines grown in 2D and 3D been used to investigate breast cancers. Breast cancer is a heterogeneous disease which causes changes to occur within the breast tissue. This includes the inability to control cell proliferation and organization, loss of cell division and the loss of cell adhesion and cell basement membrane adhesion (Imbalzano et al 2009). As a result this changes the stiffness of the tissue surrounding a tumour and can influence how the tumour cells behave. Cell lines can be used to determine how the breast tissue works and the structure of the breast tissue. MCF10A cells are almost diploid and are regarded as normal human mammary epithelial cells. It has been found that in 3D cultures these cells undergo a well-defined schedule of proliferation, differentiation and growth arrest, they form acinar structures that recap a lot of aspects of mammary architecture in vivo (Imbalzano et al 2009).

The MCF10A cell line belongs to the cell line group known as MCF10. MCF10A is thought to be normal as it does not show any traits to suggest that it is invasive, it also does not form any tumours when transplanted into immunodeficient mice (So et al 2003).

Research done in Cambridge found that by applying pressure causes tumour cells to become invasive. Within this study they used cell line MCF10A; this is the same cell line that I will be studying. The research done in Cambridge found that when cells grown in vivo in uncontrolled small spaces were exposed to pressure, it changed the stiffness of the matrix and therefore influenced the structures formed (Tsea et al 2012). This research also found that the MCF10A cells increased level of stiffness can cause MCF10A cells to have a reduced level of mechano-sensitivity, (Tsea et al 2012). Mechano-sensitivity is a specific response from cells to mechanical stimulation (Kamkin et al 2005).

3. Extracellular Matrix (ECM)

The ECM exists in all tissues and organs and is vital for the physical support of the cell; it is also responsible for recruiting essential components needed to aid morphogenesis, differentiation and homeostasis. It is mainly composed of water, proteins and polysaccharides however the ECM can differ because in each tissue of the body it is distinctively made up due to tissue growth. The ECM is responsible for cell adhesion and this is done via the ECM receptors. (Frantz et al 2010). The ECM undergoes remodelling constantly, whilst this is happening the components within the ECM are exposed to an array of modifications, this is how the ECM produces the properties intrinsic the elasticity in each organisms. The ECM also works in a protective role by preserving homeostasis and water retention (Frantz et al 2010).

The ECM is also responsible for binding growth factors and communication of receptors on the surface of cells to stimulate transduction and the regulation of gene transcription. The ECM properties are different depending on the location of each tissue and are sometimes variable in one organ for example the renal cortex and the renal medulla, both have differing ECM (Frantz et al 2010).

The ECM has two classes of macromolecules which are known as proteoglycans and fibrous proteins. Proteoglycans supply most of the extracellular interstitial space in tissues to make the gel hydrated (Frantz et al 2010). Fibrous proteins include; collagens, elastins, fibronectins and laminins. Collagen is found within the breast tissue, as an abundant fibrous protein, it is responsible for providing strength, supporting migration and directing tissue development (Frantz et al 2010).Collagen interacts with elastin. Elastin is responsible for the recoil movement in tissues which regularly stretch (Frantz et al 2010).

Fibronectin is involved in the organisation of interstitial ECM and is vital for the interaction of cell attachment and function (Frantz et al 2010).

4. 2D compared to 3D

In 100% bovine collagen the cells form ducts, in 5% matrigel/95% bovine collagen we see acini and ducts and in 50% matrigel/50% bovine collagen we get nearly all acini.

Mammalian culture cells which are grown in vitro give a clear platform for investigation of cell and tissue physiology and pathophysiology outside of an organism (Tibbitt et al 2009). Usually cultured cells are grown using single cell populations on a two-dimensional (2D) substrate; an example is tissue culture polystyrene. In vitro studies can be used to examine how epigenetic factors affect physiological occurrences, but studies have suggested that cells display unnatural behaviour when they are removed from their 3D tissue and placed into a monolayer (Miroshnikova et al 2011) .These studies have shown that when human breast tissue tumours, once removed from 2D culture and placed into 3D structure, revert back to normal growth behaviour (Miroshnikova et al 2011) .These studies have now led to much more interest in 3D scaffolds as it would appear that cells grow and react differently in 3D scaffolds, than that observed in 2D. The findings suggest that studying biological sciences in 2D is insufficient (Miroshnikova et al 2011). 3D cell culture is gaining widespread acceptance with regards to human epithelial breast tissue.

Many biologists have been investigating different types of 3D scaffolds for cell culture. An example of one of these is hydrogels, which are molecules with connecting link groups with high water content (Miroshnikova et al 2011). Hydrogels can be natural or synthetic, the growing interests in 3D cell culture has increased the demands for both types of hydrogels and also has increased interests in understanding the extra-cellular matrix (ECM) of these hydrogels to better aid in the understanding of how human breast tissue works.

5. Extracellular matrix variation

5.1 Polyacrylamide

A model often used to study the effect of ECM stiffness on cell behaviour is protein-conjugated polyacrylamide gels. These are elastic 2D gels which allow for the efficient foreseeable modulation of ECM compliance by changing cross-links concentration whilst at the same time maintaining ligand density and growth factor levels. These gels have proven useful in studying the fundamental links between ECM stiffness and cell behaviour (Miroshnikova et al 2011) .These polyacrylamide gels have been used to identify molecular methods by which ECM stiffness regulates the cell’s phenotype including emphasizing how ECM submission can regulate cell behaviour by influencing integrin adhesion and growth factor signalling (Miroshnikova et al 2011). Studies using polyacrylamide gels have shown how substantial signals from the ECM are sensed and spread, these gels have also shown how the ECM tension can change the membrane receptor function and nuclear structure to modify gene expression. The drawback of polyacrylamide gels is that they are used mainly in 2D structures but as mentioned before, cells exist in 3D format (Miroshnikova et al 2011).

A current study is underway to try and create a polyacrylamide hydrogel which may provide a gradient in mechanical stiffness of the ECM (Diederich et al 2013).

5.2 Natural and Synthetic Hydrogel’s

Natural gels include matrigel, collagen I and fibrin. These gels have been used with variable success rates to investigate the effect of ECM stiffness and topology (Miroshnikova et al 2011). Using these hydrogels stiffness has been repeatedly modulated by altering the concentration or composition of the gel. These modulations alter gel pore size, fibre architecture and the number of adhesion sites. Natural ECM’s have shown many contradictions and variations. The use of synthetic hydrogels have shown to have better control of mechanical and adhesive properties, the synthetic hydrogels have embarked on 3D designs which combine biological function and architecture of the natural gels but give stronger controls with the use of synthetic materials. An example of a synthetic gel includes agarose-stiffened collagen I gels and polyethylene glycol (PEG) (Miroshnikova et al 2011).

There are arrays of techniques used to study the use of natural and synthetic hydrogels for the use in recovering medicine some examples of hydrogels used for this include agarose, fibrin and collagen. The natural hydrogels have a similar arrangement as the extracellular matrix and are deemed biocompatible (Hesse et al 2010).

A study done in April 2011 (Turturro et al 2011) generated polyethylene glycol diacrylate (PEGDA) hydrogels and controlled the alterations such as growth factors and matrix rigidity to control and direct cell migration. This was done by using a novel photopolymerization technique and perfusion based frontal photopolymerization. This study shows that the technique used has potential in controlling the biophysical properties for direction and guidance of 3D cell behaviour (Turturro et al 2011).

5.3 Self-assembling peptides

Self-assembling peptides (SAPs), when exposed to physiological salt solutions, self-assemble into fibrils (Miroshnikova et al 2011).They are biologically compatible with biomaterials which imitate the architectural characterisation found in some natural matrices such as type I collagen gels (Miroshnikova et al 2011).SAPs are biocompatible synthetic substrates that intimate the design of natural collagen gels but they can be chemically modified. They contain 16 repeating amino acid residues that self-assemble to form nano fibres by being subjected to physiological salt conditions, they do this by forming ionic bonds between the amino acids(Miroshnikova et al 2011). A drawback for these synthetic substrates are that they do not contain ligand binding sites and are therefore unable to form ligand-dependant ECM receptor signalling, however they can be conjugated, tethered or absorbed with measurable concentrations of ligand by direct peptide conjugation or via ECM protein adsorption (Miroshnikova et al 2011) .By varying the concentrations of SAPs the stiffness of the gel can then be modified over a biological appropriate range to achieve the elasticity of soft tissue and can be used to compare the differences observed within soft tissue for example healthy breast tissue compared to cancerous breast tissue (Miroshnikova et al 2011).

SAPs support cell adhesion and can direct the separated behaviour of neural stem cells, osteoblasts, hepatocytes and endothelial cells (Miroshnikova et al 2011) .Studies have shown that they are a well-defined hydrogel system that are able to review the biochemical and micro design characterisation of the normal ECM tissue, so that the interaction between ECM accordance and multi-cellular tissue behaviour can be studied in 3D scaffolds.

SAP’s assembly has been investigated because it is thought that this may have a significant impact for developing drug delivery and regeneration targets (Rymer et al 2011).

5.4 Comparison of 3D scaffolds

There are different types of 3D cell scaffolds, examples include microporous, nanofibrous and hydrogel. There are advantages to all of these methods, microporous allow convenient enclosure of the cells but a disadvantage is that they contain porosities which have a larger cell diameter and are therefore better suited for 2D scaffolds(Tibbitt et al 2009). Nanofibrous scaffolds imitate the fibrillar ECM proteins better but a disadvantage is that they are weak with regards to stress. The disadvantages of microporous and nanofibrous scaffolds are not found within hydrogels, hydrogels better mimic the characterisation of the cell design and the mechanism of the cellular environment(Tibbitt et al 2009).

5.5 Hydrogels

Dependant on the crosslinking reaction hydrogels can be permanent hydrogels which are caused by covalent bonds. If formed by ionic or hydrogen bonding they are known as, physical hydrogels (Pal et al 2009).

Hydrogels are able to stimulate most soft tissues, because of this they are regarded as an attractive material for developing a synthetic analogue of hydrogels. Hydrogels are made up of high water content, this allows them to facilitate the transport of oxygen, nutrients and waste and allows for an elastic transport of soluble factors. Many hydrogels can be formed under mild cytocompatible conditions, (Tibbitt et al 2009) they can also be easily modified to poses cell adhesion ligands, to give them the desired elasticity and degradability(Tibbitt et al 2009).

5.6 Synthetic Hydrogels

Synthetic hydrogels often fail to take the biophysical structure of the cellular environment. It is usually necessary to engineer degradation into synthetic ECM, to try to recreate the native reorganization of the microenvironment of the cell. This is needed so that the viable cells can accumulate their own ECM, migrate and undergo morphogenesis. Synthetic hydrogels have been designed to hydrolytically decay by integrating polylactic or poly caprolactone acid units into their backbone. The number of hydrolytic bonds present usually imposes the rate of degradation; however the rate is slower than that of the normal cell process.(Tibbitt et al 2009). A recent study done (Zhu et al 2011) has found that synthetic hydrogels can be subjected to tissue engineering which can tailor structures, biodegradability and function. This can give greater controls for synthetic hydrogel’s and give better understanding to how cells work (Zhu et al 2011).

A current study which is on-going has been identifying different materials for hydrogels due to an increasing effort being made lately to create cell-encapsulated hydrogels, but the design strategies for this have not been considered. The aim of the study is to find appropriate designs for 3D cell culture and identify the challenges which may arise with the new designs (DeVolder et al 2012).

Charged gels used along with synthetic hydrogels, work better with regards to cell attachment than uncharged gels when used for cellular proliferation (Slaughter et al, 2009). Synthetic hydrogels have many advantages including the ability to be modified and being able to be produced in large-scale. The advantages of synthetic hydrogels have helped to raise the understanding of cell interactions in synthetic materials, along with an understanding of how the body reacts to unfamiliar materials. Although synthetic hydrogels have many positives, they do have a down side which includes being produced using harsh synthetic chemistry, which needs much care when synthesis is undergoing to ensure no contaminants or unreacted components are present (Slaughter et al, 2009) .

5.7 Importance of the extra-cellular Matrix (ECM)

The cellular environment has been shown to play a part to the complex signalling domain that is responsible for direct cell phenotype. A study done by Bissell has found that the phenotype can replace the efficiency of the genotype because of the interactions with the extracellular matrix (Tibbitt et al 2009). This has resulted in cells not being thought of as a single object which is defined by its genome but it must also be evaluated in the context of the extra-cellular matrix, soluble growth factors, hormones and other small molecules that regulate organism formation and function (Tibbitt et al 2009).

Integrin-binding plays an essential role not just in cell adhesion and but in most cellular processes, these binding ligands are recapped in synthetic hydrogels by the physical trapping of ECM proteins into the network, ECM proteins may include, collagen, laminin or fibronectin. These large proteins supply binding domains for integrin adhesion and have revealed improvement in cell viability and function (Tibbitt et al 2009). However trapping of these proteins can cause them to become denatured and aggregate and can also result in the introduction of multiple binding patterns and are frequently heterogeneously distributed throughout the gel all of which confound their effects (Tibbitt et al 2009).

The ECM is responsible for the intracellular cascade that influences phenotypic fates by altering gene and ultimately protein expression from that gene (Tibbitt et al 2009).

ECM including matrix structure mechanisms and dimensions has shown to have an impact on cell behaviour (Harjanto et al 2011). 3D cells have shown that they are more likely to become sterically obstructed than that of planar 2D cells. Inhibiting matrix metalloproteases (MMPs) has shown to reduce cell speed and persistence in 3D matrices resulting in the increased interest of ECM with regards to the MMP inhibitor route (Harjanto et al 2011).

6. Aims and Objectives

Aim is to see if manipulating cells or their environment alters the types of structures we normally get using the human breast cell line MCF10A, following the synthetic hydrogel route. It is hoped that there will be apparent changes in the proportion of acini to ducts using a mixture of bovine collagen and synthetic hydrogel, this can then be compared with control values obtained from 0%,5% and 50% matrigel concentrations.

Throughout the study the differences observed in gel elasticity will be monitored using rheology. If by adding hydrogel at a level which mimics the stiffness of the matrigel we get a similar proportion of acini to ducts then the effect may be largely biophysical.

To accomplish the aims set out, cells will be grown in a 3D structure, using an array of techniques including, rheology and fluorescent imaging. All methods will be done to discover whether a change in extra-cellular matrix has occurred and if so how much hydrogel was needed for this change to happen. If the stiffness does change, then the structures formed in the cultured cells will be due to differences in stiffness. Image J will be used to analyse whole mounted gels and to quantify and distinguish the different structures formed based on circularity and Feret’s diameter measurements.

7. Plan of Investigation

For my research project I will be using an array of different methods, these include: Cell culture, rheology, Fluorescent imaging and I will be imaging collagen using second harmonic imaging or reflection confocal microscopy. Below will give a brief overview of how these techniques work.

7.1 Tissue Cell Culture

Cells are spread in a synthetic gel made up of natural solutions to aid in the growth of cells. These gels are subjected to ideal growth temperature, humidity and gaseous atmosphere (Coriell Institute for Medical 2011).

7.2 Whole Mount Preparation

This will involve Carmine staining .The cells will be stained using carmine (Alum Lake) and then observed under a microscope. 1g of the carmine(Alum Lake) will be used. It is stable for up to two months and should be discarded when the colour becomes weak (Robinson 1999).

7.3 Rheometry

This technique will be used to measure the visco-elasticity of the gels. There are different types of rheometer known as rotational, capillary, or extensional. For my research project I will be using the rotational rheometer which is sometimes referred to as the stress/strain rheometer.

7.4 Fluorescent imaging

Fluorescent microscopy is a method used to obtain microscopic images of biological materials. It involves using fluorescence within a sample which is then animated with an intensely focused diffraction-limited laser beam. The focus of this beam is then scanned through the sample and the fluorescent light which has been excited is then collected via a photo-detector. This technique is usually used run with a computer which is responsible for controlling scans and recording the intensity of the fluorescence, it also is responsible for storing the images obtained from the fluorescent imaging. (Paschotta 2012)

"Fluorescent microscopy requires the use of 63X and 100X high and requires an extreme numerical aperture oil immersion which has a working distance range of 100 – 200 µm. The working distance objective is defined as the distance from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus. The working distance of the objective defines how deep into a 3D collagen matrix one can image" (Vira 2010).

7.5 Second harmonic imaging

Second harmonic imaging is based on the second harmonic generation (SHG) which is a non-linear effect that cause’s two photons from an intense laser to pass through polarisable material and are changed by the sample under investigation (Nanonics Imaging 2007).

7.6 Reflection confocal microscopy

This process works in two ways. Firstly a point on the sample is illuminated with the focus beam, the second process is attained when the conjugate focal plane of the sample is blocked by a pinhole gap, this then allows light to be released away from the sample being illuminated and blocks it from reaching the detector (LOCI 2012).

7.7 Image J

Image J is a computer based system that uses Java image processing and will aid in discovering whether a change is structure is maintained by the introduction of the synthetic hydrogel into the matrigel.

7.8 Feret’s Measurement

This will be used to measure the circularity of gels. It is regarded as the "mean distance between pairs of parallel tangents to the projected outline of the particle"(Vander 2012).