A new tumor model
Hydrogel with amphiphilic peptides is a good basis for 3D modeling of cancerous tumors
Ekaterina Gracheva, "Elements"
Fig. 1. Schematic representation of the hydrogel structure and design of the experiment. The hydrogel structure is shown at the top in the center. Orange shows the tumor cells, gray – auxiliary cells. Green circles are the attachment points of keratin molecules and peptide nanofibers. Asterisks are sections of peptides that provide interaction. The blue lines are structures consisting of peptide nanofibers and proteins. The researchers studied the composition and rigidity of the obtained hydrogels, as well as their ability to degrade. Below is a diagram of an experiment on growing cells in a hydrogel from solutions of peptides and proteins (keratin and fibronectin). The cells were placed in a solution of peptides, which was then mixed with a solution of proteins. The resulting hydrogel with cells was studied for 1-4 weeks, tracking the interaction of cells with each other and the extracellular matrix, cell growth and reaction to the effects of chemotherapeutic drugs. A drawing from the article under discussion in Science Advances.
An international team of researchers has proposed a new approach to three-dimensional modeling of cancerous tumors, which makes it possible to better reproduce their microenvironment and the conditions in which they grow in the body. As a framework for cell culture, they proposed using a hydrogel made of amphiphilic peptides assembled into long and strong nanofibers and extracellular proteins. Experiments were conducted with ovarian cancer cells. They took root well in the hydrogel and began to behave almost the same as in a real tumor. An important advantage of this approach compared to the previous ones is that the composition of the frame can be selected depending on the tumor. Scientists hope that in this way it will be possible to obtain more physiological models for different types of cancer, which will help in the search for new chemotherapeutic drugs.
Good models of malignant tumors are needed both by scientists who conduct fundamental research and by developers of new anticancer drugs. Now, most often, traditional two-dimensional cell cultures are used to model tumors: tumor cells are placed in plastic dishes, where they grow, forming a monolayer. But at the same time, in addition to the volumetric structure, many other important properties of tumors and the interactions they enter into in the body are not recreated. Volumetric cell cultures are obtained in two ways. It is possible to prevent cells from attaching to the plastic of culture dishes and creating a monolayer. This is usually achieved either by the "hanging drop" method (see R. Foty, 2011. A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids), or using dishes made of special plastic. An alternative method is to use scaffolds made of various materials to which cells can attach and create a three–dimensional structure. A detailed overview of different methods of cancer cell cultivation can be found in the article M. Kapałczyńska et al., 2018. 2D and 3D cell cultures – a comparison of different types of cancer cell cultures, and we will focus further on the "framework" methods.
The tumor consists not only of malignant cells – it includes connective tissue cells, endothelium and immune cells. All this is immersed in an extracellular matrix secreted by tumor cells, consisting mainly of structural proteins (fibronectin, collagen, keratin) and peptidoglycans (for example, hyaluronic acid). The extracellular matrix mechanically supports the cells and ensures the transport of substances between them. This set is called the tumor microenvironment. And if the presence of other types of cells in the model can be ensured by joint cultivation, then it is almost impossible to simulate all their interactions in vitro. The solution to this problem is the use of three-dimensional cultivation methods, which allows to recreate the structure and real interaction between cells.
The tumor and its microenvironment constantly "communicate" and influence each other: the microenvironment affects the growth of the tumor, and also changes itself at different stages of its development. Both the composition of cells and the composition of the extracellular matrix are changing. An ideal cell model of a tumor should reflect all these processes. Now, when creating three–dimensional cell cultures, researchers most often use Matrigel, a reagent consisting of extracellular matrix proteins and growth factors that secrete mouse tumor cells. To obtain a three-dimensional culture, tumor cells are simply added to the matrigel solution, which polymerizes under normal cultivation conditions. They aggregate, gathering into small lumps, and then divide, forming spheroids in which cells interact with each other and with the extracellular matrix – as in a real tumor, only in miniature.
Despite its popularity, matrigel has several disadvantages. Firstly, it is obtained from mouse cells, so it differs from the composition of the human extracellular matrix. Secondly, surprisingly, we still do not know its exact composition: although there have been works devoted to its detailed characterization, due to limitations of methods, it was not possible to fully determine which substances and in what quantities are included in the matrix. Moreover, the composition of the matrigel may vary slightly from batch to batch.
Because of this, scientists continue to look for other materials suitable for creating three-dimensional cellular models. There have been many attempts to use hydrogels based on hyaluronic acid, chitosan, gelatin or polyethylene glycol. Skeletons of them support the structure of the spheroid, but they lack biological activity. Therefore, there is a search for such a material that is as similar as possible to the natural extracellular matrix. An international team of researchers led by Alvaro Mata decided to use a different approach to solve this problem.
They prepared a hydrogel from two components. The first component is a keratin solution or a mixture of keratin and fibronectin solutions. There is nothing surprising in it: these proteins are present in the extracellular matrix of epithelial ovarian tumors. But the second component – amphiphilic peptides (AP) – turned out to be key to the success of the work. Being in an aqueous solution, these peptide–based molecules can independently assemble into nanofibers similar to the structure of the extracellular matrix (Fig. 2), as well as into other forms ‐ bubbles, double layers, micelles, ribbons, nanotubes (see A. Dehsorkhi et al., 2014. Self-assembling amphiphilic peptides). The properties of AP depend on the sequences of amino acids in their molecules, so it is possible to synthesize AP with different properties.
Fig. 2. Chemical structure of an amphiphilic peptide (AP). Amphiphilic peptides have a hydrophobic hydrocarbon tail (site 1) and a hydrophilic block (sites 2-5). The hydrophilic block of the molecule consists of amino acids, which are selected depending on the required properties. In this case, site 2 consists of four cysteine residues and is responsible for polymerization. Site 3 is a flexible linker site of three glycine residues. Site 4 is a phosphorylated serine residue that can interact with calcium ions, and site 5 contains the RGD (arginine–glycine–aspartate) sequence with the property of cellular adhesion. In the middle is a model of the AP molecule – it has a cone-shaped shape with a narrow hydrophobic tail and a wide hydrophilic base. Black shows carbon atoms, white – hydrogen, red – oxygen, blue – nitrogen, blue – phosphorus, yellow - sulfur. Below is a diagram of the self-assembly of AP molecules into a cylindrical structure. Illustration from the article by J. D. Hartgerink et al., 2001. Self-Assembly and Mineralization of Peptide-Amphiphile Nanofibers.
Previously, Mata's laboratory had already created AP with improved self-assembly abilities in nanofibers (which were named PA-H, see C. L. Hedegart et al., 2018. Hydrodynamically guided hierarchical self-assembly of peptide-protein bioinks). In the work under discussion, these abilities were further enhanced by adding a sequence of amino acids from elastin, an important protein of the extracellular matrix (the resulting AP molecules were called PA-VH). Additionally, the authors synthesized two auxiliary AP. One of them (PA-RGDS) potentially improves cell adhesion, and the second (PA-GHK) potentially improves cell division and can promote vascular germination into the tumor. After hyphens in the names of peptides, key motifs in hydrophilic sites are indicated (and PA is simply an abbreviation for peptide amphiphile).
The hydrogel consisted of a mixture of protein solutions and PA-VH. At the microscopic level, it consists of nanofibers assembled from AP. First, scientists tested whether this hydrogel is suitable for growing three-dimensional cell cultures. It turned out that it was suitable: it completely withstood the weight of the cells, allowing them to assemble into three-dimensional structures, and was stable enough to maintain it for a long time. At the same time, the hydrogel did not prevent the cells from secreting the extracellular matrix.
Further, the authors enclosed the cells of the ovarian epithelial tumor in the hydrogel (they do not explain why the choice was made in favor of this type of cancer; probably one of the reasons is that ovarian cancer is one of the most common types of cancer in humans, it is in the top ten of the sad list of the deadliest oncological diseases by number annual victims). The cells were placed in a keratin solution, then a PA-VH solution was added. The gel with the cells polymerized, then it was placed in a growth medium. The formation of spheroids, the interaction of cells with each other and with the matrix was observed for several weeks. At the same time, the same experiment was carried out with a matrigel as a matrix – for comparison.
The cells in the new hydrogel grew more slowly than in the matrigel, although it is rather a property of the matrigel to accelerate cell division in cultures (C. Fischbach et al., 2007. Engineering tumors with 3D scaffolds). The big plus of the new hydrogel was stability. If in the matrigel-based models, cells fell out of the spheroid on the third week, and the matrix was noticeably destroyed, then this was not observed in the models from the new hydrogel. On the 21st day of the experiment, 100% of hydrogel spheroids were preserved, and only 60% of matrigel.
Electron microscopy showed that the three-dimensional cultures in the new hydrogel are more compact, the surface has the usual structure for epithelial cells. In addition, it was seen that the cells are attached to a network of nanofibers formed by hydrogel (Fig. 3).
Fig. 3. Images of spheroids based on PA-VH hydrogel and keratin obtained using a scanning electron microscope. The age of the spheroids is 14 days. The photo on the left shows that the surface of the spheroid has a paving stone structure, common for epithelial cells. On the right, the attachment points of cells to the matrix of hydrogel nanofibers are visible. An image from the article under discussion in Science Advances.
When the tumor becomes too large, its cells no longer have enough oxygen and they initiate the germination of blood vessels. This process is triggered by malignant cells, releasing substances directed to endothelial cells, which make up the lining of blood vessels. In culture, this process can be simulated by obtaining spheroids from tumor cells and human umbilical vein endothelial cells (HUVEC). This approach is described in an article by G. Chiew et al., 2015. Physical supports from liver cancer cells are essential for differentiation and remodeling of endothelial cells in a HepG2-HUVEC co-culture model. When the spheroid grows to a sufficiently large size, HUVEC cells migrate inside it – to where there is little oxygen.
The scientists tested how HUVEC cells behave in the hydrogel under study. In three-dimensional models, they grew up, but could not establish normal interaction. One way to help them is to add extra extracellular matrix proteins to the hydrogel. Another way is to culture cells together with mesenchymal stem cells (see F. Böhrnsen, H. Schliephake, 2016. Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures). These cells secrete substances that help endothelial cells interact with each other, and subsequently contribute to the germination of blood vessels into the tumor. Therefore, the researchers combined ovarian tumor cells, HUVEC cells and human mesenchymal stem cells in one cell model. It remains to choose the right composition of the hydrogel. In addition to PA-VH, either PA-RGDS, which improves cell adhesion, or PA-GHK, which improves proliferation and can potentially promote vascular germination into the tumor, was added to the composition. Either keratin or a mixture of keratin and fibronectin were used as proteins.
When HUVEC cells interact, networks of extracellular F-actin are formed, which can be stained with the fluorescent dye phalloidin. In hydrogels with PA-GHK or PA-RGDS additives, they formed better than in those that consisted only of PA-VH. The addition of fibronectin did not affect the growth and interaction of cells. The hydrogel with the addition of PA-GHK turned out to be more brittle, therefore, a hydrogel with the addition of PA-RGDS was used for further analysis. Triple culture cells in such a hydrogel behaved the same way as in a matrigel – in this sense, the new hydrogel was not inferior to its competitor.
In the experimental hydrogel, spheroids from three cell types were denser than spheroids only from ovarian tumor cells. The authors suggest that the presence of endothelium and mesenchymal stem cells led to increased cellular interactions. The authors also observed a developed network of F-actin, including between spheroids (Fig. 4). However, it is not yet possible to draw precise conclusions that it is formed due to the migration of HUVEC cells into the spheroid and it is unclear how well it reflects the beginning of vascular germination. The authors suggest conducting additional studies and would like to optimize the composition of the hydrogel for them.
Fig. 4. Spheroids of three cell types based on hydrogel from PA-VH/PA-RGDS and keratin. On the left – immunofluorescence staining of spheroids. The red color indicates the F-actin network (phalloidin staining), green – CD31 protein (marker of endothelial cells), blue – cell nuclei (DAPI staining). On the right is a three–dimensional reconstruction of the interaction of cells. The age of the spheroids is 7 days. An image from the article under discussion in Science Advances.
Three-dimensional tumor cultures are especially needed by chemotherapy developers. The approach to their creation proposed in the work under discussion can help to more accurately simulate the behavior of chemotherapeutic drugs when injected into the body. Therefore, the authors studied how the ovarian tumor models they obtained were affected by first-line chemotherapy drugs (paclitaxel and carboplatin), as well as the matrix metalloproteinase inhibitor GM6001, which prevents the germination of blood vessels into the tumor. For this purpose, pure three-dimensional cultures of ovarian tumor cells, as well as triple models, were placed in a culture medium with chemotherapeutic drugs. In the presence of paclitaxel or carboplatin, the spheroids of both monocultures and triple cultures grew to much smaller sizes compared to untreated cells. The drug GM6001 did not affect monocultures, but somewhat reduced the growth of triple cultures, probably because it affected the extracellular matrix. Interestingly, when adding chemotherapeutic agents to three-dimensional cultures based on matrigel, the number of living cells decreased noticeably faster. This is another drawback of matrigel–based models - medicinal substances penetrate the matrix too quickly. Therefore, the results obtained on the new hydrogel should more accurately reflect what is happening in a real tumor.
The new hydrogel has another important advantage over other frameworks for three-dimensional cultures: it can be customized for different types of tumors by adding the necessary components, including growth factors. Thus, the extracellular matrix of brain tumors differs from healthy brain tissues by the presence of a large amount of hyaluronic acid and (or) collagens, as well as other content of proteoglycans chondroitin sulfates (D. Sood et al., 2019. 3D extracellular matrix microenvironment in bioengineered tissue models of primary pediatric and adult brain tumors). In the study under discussion, the authors used keratin, a protein that is contained in the extracellular matrix of the ovarian epithelium. It is also possible to change the composition and properties of amphiphilic peptides and obtain matrix nanostructures characteristic of the organ under study. In addition, such hydrogels can be used for bioprinting and the creation of artificial tissues and organs.
Source: Hedegaard et al., Peptide-protein coassembling matrices as a biomimetic 3D model of ovarian cancer // Science Advances. 2020.
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