Techniques and Technologies

Techniques and Technologies for Studying 

the ECM and Cancer

Ashley Williams

The development of appropriate techniques is one crucial component to the progression of science. Technique development is especially important in the study of human disease, where there is an urgent need to understand disease mechanisms in order to develop treatments and cures for illness.


Below are described some of the current techniques and technologies are used to study the extracellular matrix (ECM) in relation to the development of cancer. The presented methods are broken down into four broad categories: (1) cell cultures, (2) in vivo studies, (3) visualization methods, and (4) gene therapy. Within these broad categories, various techniques will be addressed including 2D vs. 3D cell cultures, the importance of studying the ECM and cancer in vivo, and staining and specialized visualization methods. To conclude, there will be a discussion on current advances in gene therapy techniques and their application to cancer and the extracellular matrix.

Cell Cultures

Cell cultures are commonly used in molecular biology in one of two forms, a 2D culture or a 3D culture (Figure 1). Each culture type has a place in biological studies and is useful as a first step in studying human diseases, such as cancer. 

Figure 1. Depiction of a 2D cell culture (left) and a 3D cell culture (right; Adjei and Blanka, 2015).

2D Cell Culture 

The majority of in vitro cell culture studies use a 2D cell culture, where cells are grown in a single layer on a petri dish (Cox and Erler, 2011). Cells grown in this way loose their natural 3D structure and have very little contact with surrounding cells. Under monolayer growth conditions, the normal levels of signalling between cells, required for actions such as apoptosis or proliferation, are diminished. Additionally, cells grown in 2D culture lack the microenvironment, including the extracellular matrix, which they are normally exposed to.

Two-dimensional analysis has been used as an initial step in studying cancer and the impact of cancer drugs; however, due to large deviations from in vivo conditions, results of 2D cell culture studies are not accurate predictors of clinical outcomes (Adjei and Blanka, 2015). Cancer cell growth in 2D assays is generally uniform and the majority of the cells are in the same phase of the cell cycle, which is not typical of in vivo cancer.

3D Cell Culture

A more clinically relevant method used to study cell growth is the scaffold-based 3D cell culture. In this type of culture, cells grow in a 3D matrix (scaffold) which mimics the ECM, simulating more in vivo-like conditions (Cox and Erler, 2011). The use of an artificial ECM allows researchers to manipulate cell behavior (ex. survival, differentiation, or migration) by changing the matrix components or stiffness. Scaffold grown cells develop a 3D shape and are able to interact with other cells on all sides, allowing for increased levels of signalling more comparable to in vivo conditions.


Three common types of scaffold-based 3D cell culture are described below (Larson, 2015):

• polymeric hard scaffolds - This type of scaffold is composed of synthetic polymers and is commonly used in (1) regenerative medicine, where the scaffold-grown cells may be used for transplantation of damaged tissues, and (2) pre-clinical testing (ex. drug testing; Figure 2). 

Figure 2. Examples of polymeric hard scaffolds (A-C; Larson, 2015).

• biological scaffolds - This type of scaffold is made from biological materials normally found within the ECM, such as fibronectin, collagen, laminin, and gelatin. Biological scaffolds provide a structural environment for cells that is closer to in vivo conditions.

• micropatterned surface microplates - Patterned plates take advantage of micro-fabrication technology where the plates are made with tiny micrometer sized compartments arranged on the bottoms of wells (Figure 3). The patterned wells allow cells to stay in greater contact with each other when compared with a traditional 2D culture. 

Figure 3. Examples of micropatterned surface microplates (A-C; Larson, 2015).

In relation to cancer research, a 3D cell culture allows researchers to better study tumor growth and response to drug treatment (Adjei and Blanka, 2015). However, while an extremely useful method, the 3D cell culture method cannot perfectly replicate a tumor's microenvironment. Consequently, cell cultures are usually used in combination with additional analysis (Cox and Erler, 2011).

For a visual of the differences between 2D and scaffold-based 3D cell cultures, check out the video below:

https://www.youtube.com/watch?v=uLOm7o8bcNw

Less pertinent to the study of the ECM are non-scaffold-based 3D cell culture, examples of which are described below (Larson, 2015):

• hanging drop microplate - Cells are grown in tiny drops and will assemble into a 3D sphere shape (Figure 4).

Figure 4. Example of a hanging drop microplate (A-B; Larson, 2015).

• spheroid microplates - Cells are grown in small v-shaped wells, allowing cells to grown in a sphere formation similar to a hanging drop plate (Figure 5).

Figure 5. Example of a spheroid microplate (A) and the shape of an individual well (B; Larson, 2015).

• microfluidic 3D cell culture - Cells are trapped in a small compartment, and fluid with nutrients and oxygen is fed in and waste is taken out each small chamber (Figure 6).

Figure 6. Example of a microfluidic 3D cell culture (Larson, 2015).

While the above described non-scaffold cell cultures may not be useful to study the ECM, application to preliminary cancer research is still valid. Despite being non-scaffold-based, hanging drop, spheroid microplate, and microfludic cell cultures provide the 3D interactions which occur in vivo. 

In vivo Studies

By far, the best way to analyze the interactions between cells and the ECM is through in vivo studies, such as with a mouse model (Figure 7; Cox and Erler, 2011). However, it is difficult to manipulate the biochemical and mechanical properties of the ECM in vivo, thus indirect approaches are often used. Examples of indirect manipulation include inhibiting expression of genes whose proteins maintain ECM structure or inducing fibrosis using irradiation or chemicals. 

Some of the techniques described later in this page are used for in vivo studies and treatments, allowing for visualization/manipulation of tumors and their microenvironment. 

Figure 7. The mouse, one of the most common mammals used in in vivo studies (Dreamatico). 

Visualization Methods

Several techniques and technologies may be used to visual the ECM and cancer. Below are listed some of the more well-known methods divided into two categories: common and specialized (Cox and Erler, 2011). Common methods are those which are used routinely to study the ECM and/or cancer, while the specialized methods tend to be more complex and have specific equipment requirements. 

Common methods

• Immunohistochemistry - This technique utilizes the interaction between antigens and antibodies to identify cellular components within their natural tissue environment (ThermoFisher). Immunohistochemistry involves three main steps: sample extraction and preparation, tissue labeling, and tissue visualization by light or fluorescent microscope. For more details on the immunohistochemistry process click here.

• Immunofluorescence - Similar to immunohistochemistry, this technique utilizes the interaction between antigens and antibodies to detect the location and relative abundance of a target protein (Davidson College). In the immunofluorescent process, a sample is incubated with a primary antibody which attaches to a target protein. The sample is then incubated with a fluorescently labeled secondary antibody which binds the primary antibody. When light hits the fluorescent label, the label absorbs the light and emits photons at a different wavelength which are detected visually. For more details on the immunofluorescence process click here. 

• Masson's trichrome - The trichrome is a three-part staining technique used to visualize cell nuclei (dark red/purple), cytoplasm (red/pink), and connective tissue (blue; Wikipedia). See below for a picture of this staining technique applied to a rat airway (Figure 8). In the context of the ECM and cancer, Masson's trichrome is used to look changes in collagen structure and/or relative abundance. 

Figure 8. Masson's trichrome staining of a rat airway (Wikipedia).

• Picrosirius red - This stain is also used to visualize collagen, specifically fibers within tissue sections embedded in paraffin (Vogel et al., 2015). The stain is red as indicated by the name and may be detected with both light and fluorescent microscopy. Below is a picture of a tumor and surrounding collagen which has been stained with picrosirius red (Figure 9). 

Figure 9. Picrosirius red staining of collagen surrounding a tumor (Apte et al., 2004).

Specialized methods

• Echocardiography - An "echo" uses ultra-high-frequency sound waves to take a live picture of the heart (American Heart Association). With echocardiography, remodeling of the extracellular matrix, specifically changes in collagen structure, which occurs after myocardial infarction may be observed (Cox and Erler, 2011). Application of the technology to cancer would allow researchers to observe extracellular changes around heart cancer cells, as well as monitor cancer metastasis to the heart (Raikhelkar et al., 2011).   
• Sonoelastography - This imaging method passes sound waves through tissues, and the resulting vibration patterns are used to generate images of the tissue (Parker, 2003). Sonoelastography is useful in visualizing not only soft tissues, but also hard tumors and the surrounding ECM within the human body.
• Second harmonic generation microscopy (SHG) - SHG uses light to visualize changes within ECM structure which occur during tumor formation (see below for more details). 

The commonly applied methods mentioned above are extremely useful; however, the techniques only give a static snapshot of the tissue being analysed (Cox and Erler, 2011). The development of specialized techniques has helped to overcome the deficits of common methods, allowing for analysis of a sample over time. In particular, second harmonic generation (SHG) microscopy, a recent innovation, is proving to be a useful tool to study the dynamic relationship between the ECM and cancer. 

SHG Imaging Microscopy

Many spectroscopy techniques, such as nonlinear optics, which have been traditionally used by chemists are now being combined with biological microscopy for the optical imaging of cells and tissues (Campagnola, 2011). At its most basic, "nonlinear optics describes the interaction of light with matter" (NLO Source). To learn more about what nonlinear optics is check out this link. 

Developing techniques involving nonlinear optics are being used in research; however, the ultimate goal is that optical microscopy will be used as a quantitative diagnostic tool for clinical applications (Campagnola, 2011). Traditional CT, MRI, and PET scans are limited to a resolution of approximately 1 mm; however, nonlinear optical techniques resolve in the µm range or smaller. A higher resolution makes optical microscopy more relevant when looking at cells which have organelles that are nanometers long. 

In traditional histological examination, tissues are removed from the organism of interest, sliced into thin sections, and then viewed from under a microscope (Campagnola, 2011). However, with emerging microscopy techniques, cells and tissues may be viewed in vivo, allowing for observation of the progression of a disease.  

SHG imaging microscopy is a type of multiphoton microscopy, which utilizes nonlinear optics (Campagnola, 2011). This type of microscopy has been used in several ECM studies, particularly for the visualization of collagen fibers.

Below are some images (Figure 10 and 11) and a video which have been taken of collagen fibers using SHG imaging microscopy. Both traditional histology methods and SHG reveal that the cancerous tissues have increased collagen and a lower cell density (Figure 10). However, SHG microscopy additionally shows the 3D changes in the fibers which cannot be see with H&E. In Figure 11, SHG microscopy was used to visualize collagen fibers in different tissues. 

Figure 10. Images of collagen fibers in the absence and presence of cancer visualized with SHG microscopy and traditional histological staining (Campagnola, 2011). 

Figure 11. Images of collagen fibers from various tissues captured by SHG microscopy (Chen et al., 2012). Images: a) tissue unspecified, b) mouse dermic, c) mouse bone, d) human ovary.

The video below is of collagen surrounding a pancreatic tumor, visualized with SHG microscopy:

www.youtube.com/watch?v=3P9Y_ArqaBo

Finally, some of the advantages of SHG microscopy over other commonly used visualization techniques are listed below (Campagnola, 2011):

1. Directly visualizes the structure of tissues unlike using dyes or colored proteins
2. Reduces photobleaching and phototoxicity compared to traditional fluorescence  
3. Visualizes tissues to depths of several hundred micrometers
4. Is non-invasive

Altogether, it appears that optical microscopy techniques such as SHG will be a large part of future cell/tissue research. The powerful nature of optical microscopy is only now being recognized within the larger biological community. As optical techniques become more popular there will likely be a drive to make the technology user-friendly, further facilitating integration.

Gene Therapy

One method of cancer treatment which has been and continues to be a part of numerous clinical trials is gene therapy (Cross and Burmester, 2006). As of 2015, 60% of all clinical trials which involved gene therapy were for cancer treatment (Yata et al., 2015). Gene therapy encompasses a wide range of treatments which all use genetic material to modify cells to cure disease (Cross and Burmester, 2006). There are three areas of gene therapy which have made it past preliminary clinical trials as described in a review by Cross and Burmester in 2006: immunotherapy, oncolytic agents, and gene transfer. Additionally, as an example application of gene therapy, results of a paper exploring bacteriophages, the ECM, and cancer will be discussed.

Immunotherapy

Immunotherapy involves boosting the immune system in order to target and destroy cancer cells (Cross and Burmester, 2006). On its own, this therapy has not been very successful; however, in combination with genetic engineering, it is more effective. 

Unlike traditional vaccines used to protect against disease, immunotherapy vaccines are used to train the body to recognize cancer cells in an effort to cure the disease or prevent progression (Cross and Burmester, 2006). To create the vaccine, cancer cells are either harvested from a patient or cell line and are grown in culture (Figure 12A). The cells are then transfected with genes which will make them more antigenic and thus more readily recognized by the immune system. Transfected cells are grown in vitro, killed, and incorporated into a vaccine.  

Immune system stimulating genes, such as those encoding cytokines, may also be introduced directly to the tumor in vivo (Figure 12B; Cross and Burmester, 2006). Once taken up by cancer cells, the gene products will cause the tumor cells to be recognized by the immune system, triggering the production of antibodies against diseased cells. 

Early immunotherapy trials in mouse cancer models were extremely promising; however, human trials have not been as successful, indicating a need for future research (Cross and Burmester, 2006). 

Figure 12. The basic steps of two methods of immunotherapy against cancer (Cross and Burmester, 2006). (A) Cancer cells are harvested, transfected with immunostimulatory genes, grown in culture, and killed. The killed cells are incorporated into a vaccine, which when administered helps the immune system recognize and target tumor cells. (B) Immune system stimulating genes are introduced directly into the tumor, where the genes produce antigenic proteins which may be recognized and destroyed by the immune system 

Oncolytic Agents

Oncolytic agents, such as a virus, are vectors used for cancer destruction (Cross and Burmester, 2006). Viruses used in this treatment are genetically engineered to target and destroy cancer cells while leaving healthy cells untouched. Oncolytic agents kill tumors through the replication of the virus, expression of toxic proteins, and/or lysis of the infected cell. Viruses used in this type of treatment (ex. adenovirus, herpes simplex virus, reovirus, etc.) are chosen based on (1) their ability to target cancers and (2) how easily they are genetically manipulated. 

The video below shows how gene transfer using an oncolytic agent may work for treating various genetic diseases:

 

www.youtube.com/watch?v=xKerTa8yLRM

An example of an oncolytic agent which has made it to clinical trials is the adenovirus, ONYX-015 (Cross and Burmester, 2006). The virus has been engineered such that it is unable to replicate within cells with functional p53 pathway activity. The absence of p53 activity, which is common in cancer cells, allows ONYX-015 to replicate within and lyse cancer cells. Trials with ONYX-015 have shown the virus to be a viable treatment for existing cancer and is being explored as a preventative measure to target cells lacking p53 activity before they proliferate out of control.

Gene Transfer

The two above methods (immunotherapy and oncolytic agents) include what is called gene transfer (Cross and Burmester, 2006). Gene transfer is the introduction of a gene into a cancer cell or the tissue surrounding the cancer cell. Genes proposed for use in cancer treatment include suicide genes which cause cell death, antiangiogenesis genes which prevent tumors from recruiting blood vessels, or genes which halt the cell cycle. A variety of vectors may be used in gene transfer treatments; however, viruses are the most common choice. Genes may also be delivered without a viral vector, such as through electroporation where an electric field is used to increase the permeability of the cell membrane. 

Gene transfer is an extremely promising therapy for cancer treatment. The technology has the potential to allow for effective, personalized treatment of not only cancer, but also many other diseases (Cross and Burmester, 2006). In the context of cancer, gene transfer has been successfully used to treat solid tumors; however, there remain many obstacles which must first be overcome before this therapy is moved beyond the clinical trial stage. Hurdles which must be jumped include increasing delivery efficiency, prevention of gene integration into non-cancer cells, and avoidance of gene silencing. 

For more information on gene therapy check out the paper "Gene Therapy for Cancer Treatment:Past, Present and Future" which provides some excellent images and descriptions of immunotherapy, oncolytic agents, and gene transfer in cancer treatment.

Bacteriophages and Cancer

Commonly, viruses which have the ability to infect humans are used in gene therapy techniques. However, in 2011, a study was published in which bacteriophages were used in conjunction with manipulation of the ECM to target and kill tumors (Figure 13). 

Figure 13. Examples of bacteriophage structures (Shmoop).

The majority of gene therapy vectors are administered via direct injection into a tumor, so consequently there is a need to develop non-invasive treatments (Yata et al., 2015). In 2015, Yata et al. used the bacteriophage adeno-associated virus/phage (AAVP) as an alternative method of gene therapy deliver. Since bacteriophages normally target bacteria and not mammalian cells, Yata et al. attached a fusion protein to the phage which would allow for targeting to receptors found only on cancer cells. 

Previously, AAVP had been used to treat naturally occurring cancers in dogs (Yata et al., 2015). With this in mind, Yata et al. sought to optimize the delivery of the phage to make treatment more efficient. To begin the optimization process, the researchers wanted to look at what impact manipulation of the ECM had on phage delivery. Normally, there is an increase of ECM density around tumors which may inhibit vectors from reaching the tumor or finding the needed receptor. Thus, decreasing ECM density before administering cancer therapeutics could potentially increase treatment efficiency, whether it be a drug or gene delivery vector.

Yata et al. manipulated the levels of ECM around tumor cells by using artificial gel matrices (2015). The samples were treated with fluorescently labeled AAVP and pictures of the samples were taken simultaneously after a designated incubation period. Figure 14 shows the results of this experiment, with red fluorescence indicative of phage internalization. Yata et al. found that that lower concentrations of ECM (2.5 mg/ml) allowed the phages to move and be internalized faster.

Figure 14. Lower ECM concentrations allow for faster movement and internalization of phages (Yata et al., 2015). Images taken simultaneously of tumor cells with different concentrations of ECM. Red fluorescence indicates the presence of the labeled phage (AAVP). 

Yata et al. also looked at whether a decrease in ECM concentration caused an increase in cancer cell killing (2015; Figure 15). To test this question, cell lines (9L, SNB19, M21, and U87) were left untreated or were treated with hyaluronidase, collagenase, or a combination of both which degraded the ECM. The treated and untreated cells were inoculated with a non-targeted AAVP (NT AAVP) or a targeted AAVP (RGD4C AAVP), both of which carried a lethal gene whose products terminate DNA synthesis. Yata et al. found that significantly more cells targeted with the RGC4C AAVP died when the ECM had been degraded compared to the untreated control. 


The results of the study of Yata et al. indicate that a decrease in ECM concentration results in enhanced RGC4C AAVP-mediated gene therapy effectiveness.

Figure 15. Reduced ECM concentration results in increased cancer cell killing with targeted AAVP therapy (RGD4C AAVP). Depicted is the percentage of survival of four different cancer cell lines (9L, SNB19, M21, and U87) with ECM degradation (red, green, yellow bars) compared with a non-degraded control (blue bar; Yata et al., 2015). Cell lines with ECM degradation were treated with a non-targeted AAVP (NT-AAVP), as well as AAVP carrying a lethal gene (RGD4C-AAVP). Results were analyzed with an ANOVA and post-hoc Tukey's; *, p<0.05; error bars are s.e.m.

The results of the study by Yata et al. help validate the ECM as a promising target to increase cancer therapy effectiveness. ECM manipulation may not only help vector-mediated delivery of genes to cancer cells, but also increase cancer drug efficiency by allowing increased amounts of drug molecules to quickly reach tumors. 

Final Comments

In the fight against cancer, useful technology and technique development has and will continue to be extremely important. As shown above, there exist many methods to study cancer and its interaction with the surrounding extracellular matrix. Analysis of the extracellular matrix and manipulation of its structure will be key in fully understanding, treating, and preventing cancer in the future.  

For more information on the ECM and cancer check out the following links:

• Purpose and structure of the ECM
• ECM and metastasis
• ECM and cancer