| BackgroundTumor is abnormal new tissue, which has a pathological change when body is under a variety of carcinogenic agents, local tissue genetically lose the normal regulation of its growth, leading its abnormal clonal proliferation. Academic circles have generally divide tumors into benign one and malignant one. Tumor, especially malignant tumor (cancer), can severely endanger the health and life of human beings. And moreover, the cancer incidence and mortality is gradually increased, it is becoming a top killer of human and one of the world’s largest public health problems in the world.Tumor microenvironment is the internal environment involved in the occurrence and development of tumor and is composed of cancer cell itself, mesenchymal cells, capillaries, tissue fluid, and a small amount of infiltrating cells. The occurrence and development of tumor is always accompanied by the interaction and regulation with the surrounding microenvironment of tumor cells. Conditions of microenvironment play a vital role on the regulation of tumorigenesis and development. Before tumor neoangiogenesis, a solid tumor forms a hypoxic microenvironment due to a rapid proliferation of malignant cells which leads to tumor progression and a poor clinical outcome. Even in a vascularized tumor, chronic ischemia and hypoxia stress induce the necrosis of cancer cells and then a necrotic core, a common feature in invasive carcinomas, appears. Mimic the microenvironment of tumor cells in vitro can provides a better approach to study mechanism of carcinogenesis, tumor cell proliferation, differentiation and clinical drug screening.So far, the cultivation of the tumor cells are using petri dishes in the 2D model and the animal model in vivo. In the 2D cultural model, tumor cell environment is obviously different from the solid tumor microenvironment in vivo, tumor cells lack of extracellular matrix (extracellular matrix, ECM), unable to interact with a variety of factors in the tumor microenvironment, it lead to cell morphogenesis, the growth, proliferation and differentiation changes which caused by the contact between cells, cells and extracellular matrix are ignored. Compared to the 2D cultural model, tumor tissue cultured in 3D have many different phenotypes of tumor cells, including proliferative cells, non-proliferative cells and necrotic cells, it can show more characteristics close to tumor cells in vivo. In addition, the animal tumor model which is established by injecting tumor cells and transplanting tumor tissue in nude mice has been recognized by most scholars, it is currently the gold standard for cancer research. However, there are some limitations in animal tumor models, tumor cells injected into nude mice will be affected by many factors in vivo, they will inevitably occur uncontrolled expression changes of gene and related protein. Therefore, it is necessary for us to explore a better method to replace the 2D culture system and animal model. Three dimensional organization construction in vitro, which make up for the inadequacy of two dimensional culture model, provides us a more efficient, economic and reliable choice to study the tumor and its related mechanism than animal model.With the rapid development of tissue engineering in recent years, the tumor tissue engineering technology based on 3D culture presents a diversified development trend. However, most of these methods are difficult to control the structure of tumors and the interaction between cells and cell matrix. For example, multicellular tumor suspension (MCTs) are composed of multiple single cells since there is no extracellular matrix between cells, and the expected interactions between cells and ECM will be lost. On the other hand, current existing 3D systems mostly rely on natural and synthetic polymers. Although natural polymers, which have properties similar to ECM, can simulate tumor microenvironment, but because of the existence of residual and impurities, experiments which use these natural materials cannot be totally controlled and absolutely rigorous. Although artificial aggregate polymeric materials avoid those defects, it lead to effective components spread and lost too quickly, which caused by its aperture between fibers can reach one thousand times or even ten thousand times the diameter of the cell. Collagen hydrogels are reported with low strength, fast degradation and their properties depending on their extraction processes, so these characteristics limit its applications. From a development perspective, all tissues should be in a hierarchical structure, which is organized from the bottom (nano or sub-nano level) to the top (macro level). Thus, the development of a nanometer scale extracellular matrix can have several advantages. First, ECM is composed of multiple types of macromolecule fibers in nanosize; second, a nanometer scale ECM has a higher surface/volume ratio to enhance cell interactions and the transport of bioactive molecules; and third, cell membrane and migration related cytoskeletons are in nano-dimensioned size. Thus, a nano-sized ECM can promote the communication between cell and cell, and cell and its milieu, and it has obvious advantages in tumorigenesis, clinical diagnosis and drug screening. Looking for suitable biological material to build three-dimensional culture tumor model via convenient method so as to reach the purpose of simulating real tumor microenvironment in vivo is the focus of this study.Gelatin and sodium alginate are natural biological materials, they have characteristics of non-toxic, biodegradable, hydrophilic, cell affinity and biocompatibility. Gelatin/sodium alginate solution can be mixed with cells to achieve cell three-dimensional culture. As a soft bracket, this tissue engineering scaffold has the advantage of containing cells not on the surface and inside the stents compared with the normal tissue engineering scaffold, it is possible for a variety of cells to achieve the reasonable distribution of space. Chitosan is a product of chitin deacetylation, belonging to the polycation polysaccharide (Pka= 6.3). N phthalein base pyran in chitosan are similar to glycosaminoglycans (GAGs) in extracellular matrix, it is biodegradable and has physiological activity. To simulate the microenvironment of tumor cells, type A gelatin, sodium alginate and molecular weight chitosan were selected to build a three dimensional multicellular tumor spheroid model in vitro via layer-by-layer assembly and cross-linking, it provides a more flexible and convenient method to study mechanism of carcinogenesis, breast cancer metastasis and drug pre-clinical evaluation.Epithelial-mesenchymal Transition (EMT) refers to the epithelial cells with polarity are induced to decreasing epithelial characteristics, and increasing mesenchymal characteristics, cell matrix adhesion disappeared and cyto skeleton remodeled under the stimulation of certain factors, it lead to epithelial cells’ polarity disappear, cell motility increase, and cells gain invasive and migration capacity. In the malignant tumor of epithelial origin, EMT is an important way to obtain the capacity of invasion and migration. Although the occurrence of EMT is closely related with the microenvironment of tumor, but the exact mechanism of tumor microenvironment inducing EMT is unclear. In this study, we compared EMT related protein expression differences of breast cancer cells between two-dimensional culture system and three-dimensional culture system, such as CD47, N-Cadherin and so on. Under the three dimensional culture system in vitro, we simulated the clinical characteristic of increased N-cadherin expression trigger EMT, it promotes tumor cell invasion and migration. We constructed that the 3D tumor model is verified.Objective1. Build a three-dimensional model for basic research and drug screening in vitro, and study the morphology, zeta potential, biocompatibility and other properties of the model.2. Constructed the validation of the three-dimensional human breast cancer tumor model in vitro.Methods1. Cell cultureThe human breast cancer cell line (MDA-MB-231) was obtained from ATCC, cells were maintained in RPMI 1640 (lower glucose, Gibco) and supplemented with 10% fetal bovine serum (FBS, Gibco),0.1% penicillin and streptomycin. Cells were maintained in an incubator (Heraeus, Germany) with 5% CO2 at 37 ℃, and the culture medium was refreshed every day. When the cell fusion rate is up to 80%, cells were digested with 0.25% trypsin solution containing 0.02% EDTA. When cells were enough, take sufficient cells for layer by layer assembly.2. LBL assembly of (gelatin-alginate)3-chitosan on single cells and its multicellular tumor spheroid formationIn order to promote the interaction between cells and cells, cells and microenvironment, we synthesis nanoscale extracellular matrix components with good compatibility. The nano scale extracellular matrix components include type A gelatin and sodium alginate, they should have opposite charge respectively. So the extracellular matrix components were deposited on the cell surface in turn. First,1 × 107 MDA-MB-231 cells were collected by centrifugation after trypsinization at 1000 rpm for 5 min. The collected MDA-MB-231 cells were gently dispersed in 7 ml of gelatin solution for 10 min, and then the gelatin solution was removed by centrifuging at 1000 rpm for 5 min. The gelatin layer was then deposited on the surface of single cells. After that, an anionic polyelectrolyte (PE) layer was deposited on top of the gelatin layer by adding 7 ml pre-warmed alginate sodium solution and gently mixed. After being incubated in an incubator for 10 min, the cationic solution was removed by centrifugation and subsequently topped by an equal volume of the cationic PE solution (type A gelatin solution). This procedure was repeated three times by the sequential deposition of oppositely charged PE layers. At the end of the deposition, a cationic polyelectrolyte (PE) layer was deposited on top of the alginate sodium layer by carefully adding 7 ml pre-warmed chitosan solution. The acidic chitosan solution was adjusted to pH 6.7 using an equal volume of RPMI 1640 medium before being used. The multicellular tumor spheroid was formed by centrifuging at 1000 rpm for 10 min and the chitosan solution was removed. The multicellular tumor spheroid was gently re-suspended in warmed RPMI 1640 medium (lower glucose), supplemented with 10% FBS and shifted to a new 6-well plate cultured in RPMI 1640 medium under a humidified atmosphere containing 5% CO2 at 37 ℃, and the culture medium was refreshed every day.3. Characterization and biocompatibility of the tissue-engineered human breast cancer three dimenssional modelAfter the building of the tissue-engineered human breast cancer three dimenssional model, the morphology, zeta potential and biocompatibility of the model and other properties were studied. In our study, optical microscope, laser particle size analyzer, scanning electron microscope, fluorescence microscope, and living/dead staining kit were used for the measurement and analysis of its properties.4. The validation of the three-dimensional human breast cancer tumor model in vitro.We compared (1) cells inside a multicellular tumor spheroid (3Dis), (2) cells migrated from the multicellular tumor spheroid (3Dms), and (3) 2D monolayer-cultured cells (2Ds) in our experiment. After four days of culture, we detected expression of p-ERK, ERK, and CD47 using western blotting after extracting total protein. Remove the multicellular tumor spheroid, washed three times with PBS, fixed with 4% paraformaldehyde for 24 hours, put in 30% sucrose solution for 1 week, and then frozen section, put it in 37℃ drying oven and baked for 24 hours, detected E-Cadherin and N-Cadherin protein expression using immunofluorescence.5. Statistical analysisAll experiments have control group. Each experiment repeated at least three times. Complete statistical analysis using SPSS 13.0 statistical software. The results were represented by mean and variance. The comparison between groups could be used single factor analysis of variance (One-way ANOVA), and applying the Tukey method in groups with homogeneity of variance, while the variance not neat, using Dunnett’s T3 method, p< 0.05 think the difference was statistically significant.Results1. The tissue-engineered anthropogenic breast cancer three-dimensional model was built successfully. It needs three cycles and a cross-linking reaction. Type A gelatin and sodium alginate were deposited on cells by electrostatic interaction. This procedure was repeated three times by the sequential deposition of oppositely charged PE layers, and then single tumor cell layer by layer assembly model obtained. At the end of the deposition, a cationic polyelectrolyte (PE) layer was deposited on top of the alginate sodium layer by carefully adding chitosan solution, the polymerization occurs between cells, and the multicellular tumor spheroid formed, namely tissue-engineered anthropogenic breast cancer three-dimensional model.2. Observation under optical microscope. The 3D multicellular tumor spheroid contains lots of cancer cells under a light microscope. Cells inside the multicellular tumor spheroid are circular and have clear outline. With the extension of time, some cells (3Dms) migrated from the multicellular tumor spheroid,3Dms and 2D cultured cells had clear outline, growed well, too.3. In order to further investigate whether cancer cells can be encapsulated in a LBL membrane, FITC conjugated gelatin was used as a LBL membrane. From the fluorescence microscope, both (gelatin-alginate)3 without chitosan and with chitosan showed that each cell was wrapped with a green fluorescence layer.4. The zeta potential of charged PE layers on cells was measured with a particle analyzer. The results showed that zeta potential presented a zigzag tendency with the sequential deposition of oppositely charged PE layers. This suggests that the surface of cell packaged on the nano materials successfully.5. The scanning electron microscope observation of the 3D multicellular tumor spheroid. The result showed that the 3D multicellular tumor spheroid had a three-dimensional structure, and nano materials were attached to the surface of cells successfully. Cells inside it grew well. There were some larger pore, the interaction between cells and cells, cells and extracellular matrix were close. It is helpful for moisture, nutrients and oxygen to permeate.6. Living/dead staining of the three dimenssional multicellular tumor spheroid. Fluorescence microscope observation shows, the entire tumor sphere is green (living cells), almost no red (dead cells). It suggests that high cell viability was maintained in the encapsulation of (gelatin-alginate)3-chitosan. Thus, the materials we used possesses good biocompatibility.7. The results of western blotting and immunofluorescence verified that the microenvironment of the 3D multicellular tumor spheroids can trigger EMT via overexpression of N-Cadherin. Western blotting analysis showed high expression of ERK and p-ERK protein in 3Dm and 2D group than that in 3Di group; and the expression of CD47 protein in the 3D multicellular tumor spheroid (3Di) was significant higher when compared with cells either cultured in the 2D cultured system (2D) or migrated from the 3D multicellular tumor spheroid (3Dm). From immunofluorescence staining, cells in 2D culture exhibited overexpression of E-cadherin around the cell membrane, but no expression of N-cadherin, and most of the cells embedded in the 3D multicellular tumor spheroid presented high expression of both N-cadherin and E-cadherin. The living cells migrated from the 3D multicellular tumor spheroid regained high expression of E-cadherin and weak expression of N-cadherin.ConclusionIn our research, we developed a 3D multicellular tumor spheroid on ultrathin matrix coated single cancer cells to mimic the extracellular milieu of solid tumor, and a series of characterization were carried out on the morphology and surface charge of the model. Cell viability was verified by live/dead cell staining assays. In this study, we also compared EMT related protein expression differences of breast cancer cells between two-dimensional culture system and three-dimensional culture system, such as CD47, N-Cadherin and so on. Under the three dimensional culture system in vitro, we simulated the clinical characteristic of increased N-cadherin expression trigger EMT, it promotes tumor cell invasion and migration. We constructed that the 3D tumor model is verified. |
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